Compositions and methods for producing circular polyribonucleotides

ABSTRACT

The present disclosure relates, generally, to compositions and methods for producing, purifying, and using circular RNA.

SEQUENCE LISTING

This application contains a Sequence Listing which has been filedelectronically in XML format and is hereby incorporated by reference inits entirety. Said XML copy, created on Feb. 8, 2023, is named51509-031003_Sequence_Listing_2_8_23.XML and is 79,410 bytes in size.

BACKGROUND

There is a need for methods of producing, purifying, and using circularpolyribonucleotides.

SUMMARY OF THE INVENTION

The disclosure provides compositions and methods for producing,purifying, and using circular RNA.

In one aspect, the invention features a linear polyribonucleotide havingthe formula 5′-(A)-(B)-(C)-(D)-(E)-(F)-(G)-3′. The linearpolyribonucleotide includes, from 5′ to 3′, (A) a 3′ half of Group Icatalytic intron fragment; (B) a 3′ splice site; (C) a 3′ exon fragment;(D) a polyribonucleotide cargo; (E) a 5′ exon fragment; (F) a 5′ splicesite; and (G) a 5′ half of Group I catalytic intron fragment. Thepolyribonucleotide includes a first annealing region that has from 2 to50, e.g., 5 to 50, e.g., 6 to 50, e.g., 7 to 50, e.g., 8 to 50 (e.g.,from 10 to 30, 10 to 20, or 10 to 15, e.g., at least 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50) ribonucleotides and is present within (A) the 3′ half ofGroup I catalytic intron fragment; (B) the 3′ splice site; or (C) the 3′exon fragment. The polyribonucleotide also includes a second annealingregion that has from 2 to 50, e.g., 5 to 50, e.g., 6 to 50, e.g., 7 to50, e.g., 8 to 50 (e.g., from 10 to 30, 10 to 20, or 10 to 15, e.g., atleast 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) ribonucleotides and is present within(E) the 5′ exon fragment; (F) the 5′ splice site; or (G) the 5′ half ofGroup I catalytic intron fragment. The first annealing region has from80% to 100% (e.g., 85% to 100%, e.g., 90% to 100%, e.g., 80%, 85%, 90%,95%, 97%, 99%, or 100%) complementarity with the second annealing regionor has from zero to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10),mismatched base pairs.

In another aspect, the invention features a linear polyribonucleotidehaving the formula 5′-(A)-(B)-(C)-(D)-(E)-(F)-(G)-3′. The linearpolyribonucleotide includes, from 5′ to 3′, (A) a 3′ half of Group Icatalytic intron fragment; (B) a 3′ splice site; (C) a 3′ exon fragment;(D) a polyribonucleotide cargo; (E) a 5′ exon fragment; (F) a 5′ splicesite; and (G) a 5′ half of Group I catalytic intron fragment, whereinthe 3′ half of Group I catalytic intron fragment of (A) and the 5′ halfof Group I catalytic intron fragment of (G) are from a CyanobacteriumAnabaena pre-tRNA-Leu gene. The polyribonucleotide includes a firstannealing region that has from 5 to 50, e.g., 6 to 50 (e.g., from 10 to30, 10 to 20, or 10 to 15, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50) ribonucleotides and is present within (A) the 3′ half of Group Icatalytic intron fragment; (B) the 3′ splice site; or (C) the 3′ exonfragment. The polyribonucleotide also includes a second annealing regionthat has from 5 to 50, e.g., 6 to 50 (e.g., from 10 to 30, 10 to 20, or10 to 15, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50)ribonucleotides and is present within (E) the 5′ exon fragment; (F) the5′ splice site; or (G) the 5′ half of Group I catalytic intron fragment.The first annealing region has from 80% to 100% (e.g., 85% to 100%,e.g., 90% to 100%, e.g., 80%, 85%, 90%, 95%, 97%, 99%, or 100%)complementarity with the second annealing region or has from zero to 10e.g., (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mismatched base pairs.

In another aspect, the invention features a linear polyribonucleotidehaving the formula 5′-(A)-(B)-(C)-(D)-(E)-(F)-(G)-3′. The linearpolyribonucleotide includes, from 5′ to 3′, (A) a 3′ half of Group Icatalytic intron fragment; (B) a 3′ splice site; (C) a 3′ exon fragment;(D) a polyribonucleotide cargo; (E) a 5′ exon fragment; (F) a 5′ splicesite; and (G) a 5′ half of Group I catalytic intron fragment, whereinthe 3′ half of Group I catalytic intron fragment of (A) and the 5′ halfof Group I catalytic intron fragment of (G) are from a Tetrahymenapre-rRNA. The polyribonucleotide includes a first annealing region thathas from 6 to 50, e.g., 7 to 50 (e.g., from 10 to 30, 10 to 20, or 10 to15, e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) ribonucleotides andis present within (A) the 3′ half of Group I catalytic intron fragment;(B) the 3′ splice site; or (C) the 3′ exon fragment. Thepolyribonucleotide also includes a second annealing region that has from6 to 50, e.g., 7 to 50 (e.g., from 10 to 30, 10 to 20, or 10 to 15,e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) ribonucleotides and ispresent within (E) the 5′ exon fragment; (F) the 5′ splice site; or (G)the 5′ half of Group I catalytic intron fragment. The first annealingregion has from 80% to 100% (e.g., 85% to 100%, e.g., 90% to 100%, e.g.,80%, 85%, 90%, 95%, 97%, 99%, or 100%) complementarity with the secondannealing region or has from zero to 10 e.g., (0, 1, 2, 3, 4, 5, 6, 7,8, 9, or 10) mismatched base pairs.

In some embodiments, (A) or (C) includes the first annealing region and(E) or (G) includes the second annealing region.

In some embodiments, the 3′ exon fragment of (C) includes the firstannealing region and the 5′ exon fragment of (E) includes the secondannealing region.

In some embodiments, the 3′ exon fragment of (C) includes the firstannealing region and the 5′ half of Group I catalytic intron fragment of(G) includes the second annealing region.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) includes the first annealing region and the 5′ exon fragment of (E)includes the second annealing region.

In some embodiments, first annealing region and the second annealingregion include zero or one mismatched base pair.

In some embodiments, the first annealing region and the second annealingregion are 100% complementary.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ half of Group I catalytic intron fragment of (G) are froma cyanobacterium Anabaena pre-tRNA-Leu gene, a Tetrahymena pre-rRNA, ora T4 phage td gene.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ half of Group I catalytic intron fragment of (G) are froma Cyanobacterium Anabaena pre-tRNA-Leu gene, and the 3′ exon fragment of(C) includes the first annealing region and the 5′ exon fragment of (E)includes the second annealing region. The first annealing region mayinclude, e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11, 12, 13,14, or 15) ribonucleotides and the second annealing region may include,e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11, 12, 13, 14, or15) ribonucleotides.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ half of Group I catalytic intron fragment of (G) are froma Tetrahymena pre-rRNA, and the 3′ half of Group I catalytic intronfragment of (A) includes the first annealing region and the 5′ exonfragment of (E) includes the second annealing region. In someembodiments, the 3′ exon fragment of (C) includes the first annealingregion and the 5′ half of Group I catalytic intron fragment of (G)includes the second annealing region. The first annealing region mayinclude, e.g., from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11, 12, 13,14, 15, or 16) ribonucleotides, and the second annealing region mayinclude, e.g., from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11, 12, 13,14, 15, or 16) ribonucleotides.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ Group I catalytic intron fragment of (G) are from a T4phage td gene. The 3′ exon fragment of (C) may include the firstannealing region and the 5′ half of Group I catalytic intron fragment of(G) may include the second annealing region. The first annealing regionmay include, e.g., from 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the secondannealing region may include, e.g., from 2 to 16, e.g., 10 to 16 (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) is the 5′ terminus of the linear polynucleotide.

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) is the 3′ terminus of the linear polyribonucleotide.

In some embodiments, the linear polyribonucleotide does not include afurther annealing region.

In some embodiments, the linear polyribonucleotide does not include anannealing region 3′ to (A) that includes partial or complete nucleicacid complementarity with an annealing region 5′ to (G).

In some embodiments, the polyribonucleotide cargo of (D) includes anexpression sequence, a non-coding sequence, or an expression sequenceand a non-coding sequence.

In some embodiments, the polyribonucleotide cargo of (D) includes anexpression sequence encoding a polypeptide.

In some embodiments, the polyribonucleotide cargo of (D) includes anIRES operably linked to an expression sequence encoding a polypeptide.

In some embodiments, the IRES is located upstream of the expressionsequence. In some embodiments, the IRES is located downstream of theexpression sequence.

In some embodiments, the polyribonucleotide cargo of (D) includes anexpression sequence that encodes a polypeptide that has a biologicaleffect on a subject.

In some embodiments, the linear polyribonucleotide further includes afirst spacer region between the 3′ exon fragment of (C) and thepolyribonucleotide cargo of (D). The first spacer region may be, e.g.,at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotidesin length. In some embodiments, the linear polyribonucleotide furtherincludes a second spacer region between the polyribonucleotide cargo of(D) and the 5′ exon fragment of (E). The second spacer region may be,e.g., at least 5 (e.g., at least 10, at least 15, at least 20)ribonucleotides in length. In some embodiments, each spacer region is atleast 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides inlength. Each spacer region may be, e.g., from 5 to 500 (e.g., 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or500) ribonucleotides in length. The first spacer region, the secondspacer region, or the first spacer region and the second spacer regionmay include a polyA sequence. The first spacer region, the second spacerregion, or the first spacer region and the second spacer region mayinclude a polyA-C sequence. The first spacer region, the second spacerregion, or the first spacer region and the second spacer region mayinclude a polyA-G sequence. The first spacer region, the second spacerregion, or the first spacer region and the second spacer region mayinclude a polyA-T sequence. The first spacer region, the second spacerregion, or the first spacer region and the second spacer region mayinclude a random sequence.

In some embodiments, the linear polyribonucleotide is from 50 to 20,000,e.g., 100 to 20,000, e.g., 200 to 20,000, e.g., 300 to 20,000 (e.g., 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500,4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000,13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000)ribonucleotides in length. In embodiments, the linear polyribonucleotideis, e.g., at least 50, at least 100, at least 200, at least 300, atleast 400, at least 500, at least 1,000, at least 2,000, at least 3,000,at least 4,000, or at least 5,000 ribonucleotides in length.

In another aspect, the invention features a DNA vector including an RNApolymerase promoter operably linked to a DNA sequence that encodes thelinear polyribonucleotide of any of the embodiments described herein.

In another aspect, the invention features a circular polyribonucleotide(e.g., a covalently closed circular polyribonucleotide) produced fromthe linear polyribonucleotide or the DNA vector of any of theembodiments described herein.

In another aspect, the invention features a circular polyribonucleotide(e.g., a covalently closed circular polyribonucleotide) having a splicejunction joining a 5′ exon fragment and a 3′ exon fragment. The 3′ exonfragment includes a first annealing region including 2 to 50, e.g., 5 to50, e.g., 6 to 50, e.g., 7 to 50, e.g., 8 to 50 (e.g., from 10 to 30, 10to 20, or 10 to 15, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) ribonucleotides,and the 5′ exon fragment includes a second annealing region including 2to 50, e.g., 5 to 50, e.g., 6 to 50, e.g., 7 to 50, e.g., 8 to 50 (e.g.,from 10 to 30, 10 to 20, or 10 to 15, e.g., 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50) ribonucleotides. In embodiment, the first annealing region and thesecond annealing region include from 80% to 100% (e.g., 80%, 85%, 90%,95%, 97%, 99%, or 100%) complementarity. In embodiments the firstannealing region and the second annealing region include from zero to 10(e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mismatched base pairs (bp).In embodiments, the circular polynucleotide further include apolyribonucleotide cargo. In embodiments, the polyribonucleotide cargoincludes an expression (or coding) sequence, a non-coding sequence, or acombination of an expression (or coding) sequence and a non-codingsequence. In embodiments, the polyribonucleotide cargo includes anexpression (coding) sequence encoding a polypeptide. In embodiments, thepolyribonucleotide includes an IRES operably linked to an expressionsequence encoding a polypeptide. In some embodiments, the circularpolyribonucleotide further includes a spacer region between the IRES andthe 3′ exon fragment or the 5′ exon fragment. The spacer region may be,e.g., at least 5 (e.g., at least 10, at least 15, at least 20)ribonucleotides in length ribonucleotides in length. The spacer regionmay be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides. In someembodiments, the spacer region includes a polyA sequence. In someembodiments, the spacer region includes a polyA-C sequence. In someembodiments, the spacer region includes a polyA-G sequence. In someembodiments, the spacer region includes a polyA-T sequence. In someembodiments, the spacer region includes a random sequence.

In some embodiments, the circular polyribonucleotide is from 50 to20,000, e.g., 100 to 20,000, e.g., 200 to 20,000, e.g., 300 to 20,000(e.g., 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,3,000, 3,500, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000,12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or20,000) ribonucleotides in length. In embodiments, the circularpolyribonucleotide is, e.g., at least 500, at least 1,000, at least2,000, at least 3,000, at least 4,000, or at least 5,000 ribonucleotidesin length.

In some embodiments, the circular polyribonucleotide is produced from alinear polyribonucleotide or vector as described herein.

In another aspect, the invention features a method of expressing apolypeptide in a cell by providing a linear polyribonucleotide, a DNAvector, or a circular polyribonucleotide as described herein to thecell. The method further includes allowing the cellular machinery toexpress the polypeptide from the polyribonucleotide.

In another aspect, the invention features a method of producing acircular polyribonucleotide as described herein by providing a linearpolyribonucleotide as described herein under conditions suitable forself-splicing of the linear polyribonucleotide to produce the circularpolyribonucleotide.

DEFINITIONS

To facilitate the understanding of this disclosure, a number of termsare defined below. Terms defined herein have meanings as commonlyunderstood by a person of ordinary skill in the areas relevant to thedisclosure. Terms such as “a”, “an,” and “the” are not intended to referto only a singular entity but include the general class of which aspecific example may be used for illustration. The term “or” is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternative are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and “and/or”. Theterminology herein is used to describe specific embodiments, but theirusage is not to be taken as limiting, except as outlined in the claims.

As used herein, any values provided in a range of values include boththe upper and lower bounds, and any values contained within the upperand lower bounds.

As used herein, the term “about” refers to a value that is within ± 10%of a recited value.

As used herein, the term “carrier” is a compound, composition, reagent,or molecule that facilitates the transport or delivery of a composition(e.g., a circular polyribonucleotide) into a cell by a covalentmodification of the circular polyribonucleotide, via a partially orcompletely encapsulating agent, or a combination thereof. Non-limitingexamples of carriers include carbohydrate carriers (e.g., ananhydride-modified phytoglycogen or glycogen-type material),nanoparticles (e.g., a nanoparticle that encapsulates or is covalentlylinked binds to the circular polyribonucleotide), liposomes, fusosomes,ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g.,a protein covalently linked to the circular polyribonucleotide), orcationic carriers (e.g., a cationic lipopolymer or transfectionreagent).

As used herein, the terms “circular polyribonucleotide” and “circularRNA” are used interchangeably and mean a polyribonucleotide moleculethat has a structure having no free ends (i.e., no free 3′ or 5′ ends),for example a polyribonucleotide molecule that forms a circular orend-less structure through covalent or non-covalent bonds. The circularpolyribonucleotide may be, e.g., a covalently closed polyribonucleotide.

As used herein, the term “circularization efficiency” is a measurementof resultant circular polyribonucleotide versus its non-circularstarting material.

As used herein, the terms “disease,” “disorder,” and “condition” eachrefer to a state of sub-optimal health, for example, a state that is orwould typically be diagnosed or treated by a medical professional.

By “heterologous” is meant to occur in a context other than in thenaturally occurring (native) context. A “heterologous” polynucleotidesequence indicates that the polynucleotide sequence is being used in away other than what is found in that sequence’s native genome. Forexample, a “heterologous promoter” is used to drive transcription of asequence that is not one that is natively transcribed by that promoter;thus, a “heterologous promoter” sequence is often included in anexpression construct by means of recombinant nucleic acid techniques.The term “heterologous” is also used to refer to a given sequence thatis placed in a non-naturally occurring relationship to another sequence;for example, a heterologous coding or non-coding nucleotide sequence iscommonly inserted into a genome by genomic transformation techniques,resulting in a genetically modified or recombinant genome.

As used herein “increasing fitness” or “promoting fitness” of a subjectrefers to any favorable alteration in physiology, or of any activitycarried out by a subject organism, as a consequence of administration ofa peptide or polypeptide described herein, including, but not limitedto, any one or more of the following desired effects: (1) increasedtolerance of biotic or abiotic stress by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) increased yield orbiomass by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,100% or more; (3) modified flowering time by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increasedresistance to pests or pathogens by about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99%, 100% or more, (4) increased resistance toherbicides by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or more; (5) increasing a population of a subject organism(e.g., an agriculturally important insect) by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (6) increasing thereproductive rate of a subject organism (e.g., insect, e.g., bee orsilkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or more; (7) increasing the mobility of a subject organism(e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8) increasing the bodyweight of a subject organism (e.g., insect, e.g., bee or silkworm) byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% ormore; (9) increasing the metabolic rate or activity of a subjectorganism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (10) increasingpollination (e.g., number of plants pollinated in a given amount oftime) by a subject organism (e.g., insect, e.g., bee or silkworm) byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% ormore; (11) increasing production of subject organism (e.g., insect,e.g., bee or silkworm) byproducts (e.g., honey from a honeybee or silkfrom a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, 100% or more; (12) increasing nutrient content of the subjectorganism (e.g., insect) (e.g., protein, fatty acids, or amino acids) byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% ormore; or (13) increasing a subject organism’s resistance to pesticides(e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorusinsecticide (e.g., a phosphorothioate, e.g., fenitrothion)) by about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more,(14) increasing health or reducing disease of a subject organism such asa human or non-human animal. An increase in host fitness can bedetermined in comparison to a subject organism to which the modulatingagent has not been administered. Conversely, “decreasing fitness” of asubject refers to any unfavorable alteration in physiology, or of anyactivity carried out by a subject organism, as a consequence ofadministration of a peptide or polypeptide described herein, including,but not limited to, any one or more of the following intended effects:(1) decreased tolerance of biotic or abiotic stress by about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) decreasedyield or biomass by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, 100% or more; (3) modified flowering time by about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) decreasedresistance to pests or pathogens by about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99%, 100% or more, (4) decreased resistance toherbicides by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or more; (5) decreasing a population of a subject organism(e.g., an agriculturally important insect) by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (6) decreasing thereproductive rate of a subject organism (e.g., insect, e.g., bee orsilkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or more; (7) decreasing the mobility of a subject organism(e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8) decreasing the bodyweight of a subject organism (e.g., insect, e.g., bee or silkworm) byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% ormore; (9) decreasing the metabolic rate or activity of a subjectorganism (e.g., insect, e.g., bee or silkworm) by about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (10) decreasingpollination (e.g., number of plants pollinated in a given amount oftime) by a subject organism (e.g., insect, e.g., bee or silkworm) byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% ormore; (11) decreasing production of subject organism (e.g., insect,e.g., bee or silkworm) byproducts (e.g., honey from a honeybee or silkfrom a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, 100% or more; (12) decreasing nutrient content of the subjectorganism (e.g., insect) (e.g., protein, fatty acids, or amino acids) byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% ormore; or (13) decreasing a subject organism’s resistance to pesticides(e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorusinsecticide (e.g., a phosphorothioate, e.g., fenitrothion)) by about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more,(14) decreasing health or reducing disease of a subject organism such asa human or non-human animal. A decrease in host fitness can bedetermined in comparison to a subject organism to which the modulatingagent has not been administered. It will be apparent to one of skill inthe art that certain changes in the physiology, phenotype, or activityof a subject, e.g., modification of flowering time in a plant, can beconsidered to increase fitness of the subject or to decrease fitness ofthe subject, depending on the context (e.g., to adapt to a change inclimate or other environmental conditions). For example, a delay inflowering time (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, 100% fewer plants in a population flowering at a givencalendar date) can be a beneficial adaptation to later or coolerspringtimes and thus be considered to increase a plant’s fitness;conversely, the same delay in flowering time in the context of earlieror warmer springtimes can be considered to decrease a plant’s fitness.

As used herein, the terms “linear RNA” or “linear polyribonucleotide” or“linear polyribonucleotide molecule” are used interchangeably and meanpolyribonucleotide molecule having a 5′ and 3′ end. One or both of the5′ and 3′ ends may be free ends or joined to another moiety. Linear RNAincludes RNA that has not undergone circularization (e.g., ispre-circularized) and can be used as a starting material forcircularization.

As used herein, the term “modified ribonucleotide” means a nucleotidewith at least one modification to the sugar, the nucleobase, or theinternucleoside linkage.

As used herein, the term “naked delivery” is a formulation for deliveryto a cell without the aid of a carrier and without covalent modificationto a moiety that aids in delivery to a cell. A naked deliveryformulation is free from any transfection reagents, cationic carriers,carbohydrate carriers, nanoparticle carriers, or protein carriers. Forexample, naked delivery formulation of a circular polyribonucleotide isa formulation that comprises a circular polyribonucleotide withoutcovalent modification and is free from a carrier.

The term “pharmaceutical composition” is intended to also disclose thatthe circular or linear polyribonucleotide included within apharmaceutical composition can be used for the treatment of the human oranimal body by therapy.

The term “polynucleotide” as used herein means a molecule including oneor more nucleic acid subunits, or nucleotides, and can be usedinterchangeably with “nucleic acid” or “oligonucleotide”. Apolynucleotide can include one or more nucleotides selected fromadenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), orvariants thereof. A nucleotide can include a nucleoside and at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotidecan include a nucleobase, a five-carbon sugar (either ribose ordeoxyribose), and one or more phosphate groups. Ribonucleotides arenucleotides in which the sugar is ribose. Polyribonucleotides orribonucleic acids, or RNA, can refer to macromolecules that includemultiple ribonucleotides that are polymerized via phosphodiester bonds.Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.As used herein, a polyribonucleotide sequence that recites thymine (T)is understood to represent uracil (U).

As used herein, the term “polyribonucleotide cargo” herein includes anysequence including at least one polyribonucleotide. In embodiments, thepolyribonucleotide cargo includes one or multiple expression sequences,wherein each expression sequence encodes a polypeptide. In embodiments,the polyribonucleotide cargo includes one or multiple noncodingsequences, such as a polyribonucleotide having regulatory or catalyticfunctions. In embodiments, the polyribonucleotide cargo includes acombination of expression and noncoding sequences. In embodiments, thepolyribonucleotide cargo includes one or more polyribonucleotidesequence described herein, such as one or multiple regulatory elements,internal ribosomal entry site (IRES) elements, or spacer sequences.

As used interchangeably herein, the terms “polyA” or “polyA sequence”refer to an untranslated, contiguous region of a nucleic acid moleculeof at least 5 nucleotides in length and consisting of adenosineresidues. In some embodiments, a polyA sequence is at least 10, at least15, at least 20, at least 30, at least 40, or at least 50 nucleotides inlength. In some embodiments, a polyA sequence is located 3′ to (e.g.,downstream of) an open reason frame (e.g., an open reading frameencoding a polypeptide), and the polyA sequence is 3′ to a terminationelement (e.g., a Stop codon) such that the polyA is not translated. Insome embodiments, a polyA sequence is located 3′ to a terminationelement and a 3′ untranslated region.

As used herein, the elements of a nucleic acid are “operably connected”if they are positioned on the vector such that they can be transcribedto form a linear RNA that can then be circularized into a circular RNAusing the methods provided herein.

Polydeoxyribonucleotides or deoxyribonucleic acids, or DNA, meansmacromolecules that include multiple deoxyribonucleotides that arepolymerized via phosphodiester bonds. A nucleotide can be a nucleosidemonophosphate or a nucleoside polyphosphate. A nucleotide means adeoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleosidetriphosphate (dNTP), which can be selected from deoxyadenosinetriphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosinetriphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidinetriphosphate (dTTP) dNTPs, that include detectable tags, such asluminescent tags or markers (e.g., fluorophores). A nucleotide caninclude any subunit that can be incorporated into a growing nucleic acidstrand. Such subunit can be an A, C, G, T, or U, or any other subunitthat is specific to one or more complementary A, C, G, T or U, orcomplementary to a purine (i.e., A or G, or variant thereof) or apyrimidine (i.e., C, T or U, or variant thereof). In some examples, apolynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA),or derivatives or variants thereof. In some cases, a polynucleotide is ashort interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA),a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA(mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), to name a few,and encompasses both the nucleotide sequence and any structuralembodiments thereof, such as single-stranded, double-stranded,triple-stranded, helical, hairpin, etc. In some cases, a polynucleotidemolecule is circular. A polynucleotide can have various lengths. Anucleic acid molecule can have a length of at least about 10 bases, 20bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases,400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb,50 kb, or more. A polynucleotide can be isolated from a cell or atissue. Embodiments of polynucleotides include isolated and purifiedDNA/RNA molecules, synthetic DNA/RNA molecules, and synthetic DNA/RNAanalogs.

Embodiments of polynucleotides, e.g., polyribonucleotides orpolydeoxyribonucleotides, include polynucleotides that contain one ormore nucleotide variants, including nonstandard nucleotide(s),non-natural nucleotide(s), nucleotide analog(s) or modified nucleotides.Examples of modified nucleotides include, but are not limited todiaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta—D— mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acidmethylester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil,3-(3-amino- 3- N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine andthe like. In some cases, nucleotides include modifications in theirphosphate moieties, including modifications to a triphosphate moiety.Non-limiting examples of such modifications include phosphate chains ofgreater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 ormore phosphate moieties) and modifications with thiol moieties (e.g.,alpha-thiotriphosphate and beta-thiotriphosphates). In embodiments,nucleic acid molecules are modified at the base moiety (e.g., at one ormore atoms that typically are available to form a hydrogen bond with acomplementary nucleotide or at one or more atoms that are not typicallycapable of forming a hydrogen bond with a complementary nucleotide),sugar moiety or phosphate backbone. In embodiments, nucleic acidmolecules contain amine -modified groups, such as amino allyl 1-dUTP(aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP) to allow covalentattachment of amine reactive moieties, such as N-hydroxysuccinimideesters (NHS). Alternatives to standard DNA base pairs or RNA base pairsin the oligonucleotides of the present disclosure can provide higherdensity in bits per cubic mm, higher safety (resistant to accidental orpurposeful synthesis of natural toxins), easier discrimination inphoto-programmed polymerases, or lower secondary structure. Suchalternative base pairs compatible with natural and mutant polymerasesfor de novo or amplification synthesis are described in Betz K, MalyshevDA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P,Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, which isherein incorporated by reference for all purposes.

As used herein, “polypeptide” means a polymer of amino acid residues(natural or unnatural) linked together most often by peptide bonds. Theterm, as used herein, refers to proteins, polypeptides, and peptides ofany size, structure, or function. Polypeptides can include geneproducts, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide can be a singlemolecule or a multi-molecular complex such as a dimer, trimer, ortetramer. They can also include single chain or multichain polypeptidessuch as antibodies or insulin and can be associated or linked. Mostcommonly disulfide linkages are found in multichain polypeptides. Theterm polypeptide can also apply to amino acid polymers in which one ormore amino acid residues are an artificial chemical analogue of acorresponding naturally occurring amino acid.

As used herein, the term “plant-modifying polypeptide” refers to apolypeptide that can alter the genetic properties (e.g., increase geneexpression, decrease gene expression, or otherwise alter the nucleotidesequence of DNA or RNA), epigenetic properties, or biochemical orphysiological properties of a plant in a manner that results in a changein the plant’s physiology or phenotype, e.g., an increase or a decreasein plant fitness.

As used herein, the term “regulatory element” is a moiety, such as anucleic acid sequence, that modifies expression of an expressionsequence within the circular or linear polyribonucleotide.

As used herein, a “spacer” refers to any contiguous nucleotide sequence(e.g., of one or more nucleotides) that provides distance or flexibilitybetween two adjacent polynucleotide regions.

As used herein, the term “sequence identity” is determined by alignmentof two peptide or two nucleotide sequences using a global or localalignment algorithm. Sequences are referred to as “substantiallyidentical” or “essentially similar” when they share at least a certainminimal percentage of sequence identity when optimally aligned (e.g.,when aligned by programs such as GAP or BESTFIT using defaultparameters). GAP uses the Needleman and Wunsch global alignmentalgorithm to align two sequences over their entire length, maximizingthe number of matches and minimizes the number of gaps. Generally, theGAP default parameters are used, with a gap creation penalty = 50(nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides)/ 2 (proteins). For nucleotides the default scoring matrix used isnwsgapdna, and for proteins the default scoring matrix is Blosum62(Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments andscores for percentage sequence identity are determined, e.g., usingcomputer programs, such as the GCG Wisconsin Package, Version 10.3,available from Accelrys Inc., 9685 Scranton Road, San Diego, CA92121-3752 USA, or EmbossWin version 2.10.0 (using the program“needle”). Alternatively or additionally, percent identity is determinedby searching against databases, e.g., using algorithms such as FASTA,BLAST, etc. Sequence identity refers to the sequence identity over theentire length of the sequence.

As used herein, “structured” with regard to RNA refers to an RNAsequence that is predicted by the RNAFold software or similar predictivetools to form a structure (e.g., a hairpin loop) with itself or othersequences in the same RNA molecule.

As used herein, the term “subject” refers to an organism, such as ananimal, plant, or microbe. In embodiments, the subject is a vertebrateanimal (e.g., mammal, bird, fish, reptile, or amphibian). Inembodiments, the subject is a human. In embodiments, the subject is anon-human mammal. In embodiments, the subject is a non-human mammal suchas a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle,buffalo, bison, sheep, goat, pig, camel, llama, alpaca, deer, horses,donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), orlagomorph (e.g., rabbit). In embodiments, the subject is a bird, such asa member of the avian taxa Galliformes (e.g., chickens, turkeys,pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae(e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), orPsittaciformes (e.g., parrots). In embodiments, the subject is aninvertebrate such as an arthropod (e.g, insects, arachnids,crustaceans), a nematode, an annelid, a helminth, or a mollusc. Inembodiments, the subject is an invertebrate agricultural pest or aninvertebrate that is parasitic on an invertebrate or vertebrate host. Inembodiments, the subject is a plant, such as an angiosperm plant (whichcan be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, acycad, a gnetophyte, a Ginkgo), a fern, horsetail, clubmoss, or abryophyte. In embodiments, the subject is a eukaryotic alga (unicellularor multicellular). In embodiments, the subject is a plant ofagricultural or horticultural importance, such as row crop plants,fruit-producing plants and trees, vegetables, trees, and ornamentalplants including ornamental flowers, shrubs, trees, groundcovers, andturf grasses.

As used herein, the term “treat,” or “treating,” refers to aprophylactic or therapeutic treatment of a disease or disorder (e.g., aninfectious disease, a cancer, a toxicity, or an allergic reaction) in asubject. The effect of treatment can include reversing, alleviating,reducing severity of, curing, inhibiting the progression of, reducingthe likelihood of recurrence of the disease or one or more symptoms ormanifestations of the disease or disorder, stabilizing (i.e., notworsening) the state of the disease or disorder, or preventing thespread of the disease or disorder as compared to the state or thecondition of the disease or disorder in the absence of the therapeutictreatment. Embodiments include treating plants to control a disease oradverse condition caused by or associated with an invertebrate pest or amicrobial (e.g., bacterial, fungal, oomycete, or viral) pathogen.Embodiments include treating a plant to increase the plant’s innatedefense or immune capability to tolerate pest or pathogen pressure.

As used herein, the term “termination element” is a moiety, such as anucleic acid sequence, that terminates translation of the expressionsequence in the circular or linear polyribonucleotide.

As used herein, the term “translation efficiency” is a rate or amount ofprotein or peptide production from a ribonucleotide transcript. In someembodiments, translation efficiency can be expressed as amount ofprotein or peptide produced per given amount of transcript that codesfor the protein or peptide, e.g., in a given period of time, e.g., in agiven translation system, e.g., an cell-free translation system likerabbit reticulocyte lysate.

As used herein, the term “translation initiation sequence” is a nucleicacid sequence that initiates translation of an expression sequence inthe circular or linear polyribonucleotide.

As used herein, the term “therapeutic polypeptide” refers to apolypeptide that when administered to or expressed in a subject providessome therapeutic benefit. In embodiments, a therapeutic polypeptide isused to treat or prevent a disease, disorder, or condition in a subjectby administration of the therapeutic peptide to a subject or byexpression in a subject of the therapeutic polypeptide. In alternativeembodiments, a therapeutic polypeptide is expressed in a cell and thecell is administered to a subject to provide a therapeutic benefit.

As used herein, a “vector” means a piece of DNA, that is synthesized(e.g., using PCR), or that is taken from a virus, plasmid, or cell of ahigher organism into which a foreign DNA fragment can be or has beeninserted for cloning or expression purposes. In some embodiments, avector can be stably maintained in an organism. A vector can include,for example, an origin of replication, a selectable marker or reportergene, such as antibiotic resistance or GFP, or a multiple cloning site(MCS). The term includes linear DNA fragments (e.g., PCR products,linearized plasmid fragments), plasmid vectors, viral vectors, cosmids,bacterial artificial chromosomes (BACs), yeast artificial chromosomes(YACs), and the like. In one embodiment, the vectors provided hereininclude a multiple cloning site (MCS). In another embodiment, thevectors provided herein do not include an MCS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic drawings showing an exemplary Anabaenapermuted intron-exon with an annealing region of 5 nucleotides (FIG. 1A)and an exemplary Anabaena permuted intron-exon with an extendedannealing region (FIG. 1B).

FIGS. 2A and 2B are schematic drawings showing the structures of anexemplary Anabaena permuted intron-exon with an annealing region of 5nucleotides (FIG. 2A) and an exemplary Anabaena permuted intron-exonwith an extended annealing region (FIG. 2B)

FIGS. 3A and 3B are graphs showing the circularization efficiency ofAnabaena permuted intron-exon with an annealing region of 5 nucleotides(Anabaena 1), Anabaena permuted intron-exon with an extended annealingregion (Anabaena 2), and Anabaena 3 with either a 1.2 Kb RNA (FIG. 3A)or a 4.5 Kb RNA (FIG. 3B).

FIG. 4 is a graph showing relative expression of Gluc from circular RNAgenerated with Anabaena permuted intron-exon with an annealing region of5 nucleotides (Anabaena 1), Anabaena permuted intron-exon with anextended annealing region (Anabaena 2), or Anabaena 3 at three differenttimepoints.

FIG. 5 is a graph showing relative expression SARS-CoV-2 spike proteinfrom circular RNA generated with Anabaena permuted intron-exon with anannealing region of 5 nucleotides (Anabaena 1), Anabaena permutedintron-exon with an extended annealing region (Anabaena 2), or Anabaena3 at three different timepoints.

FIG. 6 is a schematic drawing showing exemplary designs of Anabaenapermuted intron-exon with several extended annealing regions between E2and E1.

FIG. 7 is a graph showing circularization efficiency with the Anabaenapermuted intron-exon with an extended annealing region (Anabaena 2), andAnabaena permuted intron-exon with further 5, 10, or 15 nucleotideextensions of the annealing region.

FIG. 8 is a graph showing expression with the Anabaena permutedintron-exon with an extended annealing region (Anabaena 2), and Anabaenapermuted intron-exon with further 5, 10, or 15 nucleotide extensions ofthe annealing region at three different timepoints.

FIGS. 9A and 9B are schematic drawings showing an exemplary Tetrahymenapermuted intron-exon with an annealing region of 6 nucleotides (FIG. 9A)and an exemplary Tetrahymena permuted intron-exon with an extendedannealing region (FIG. 9B).

FIGS. 10A and 10B are schematic drawings showing the structures of anexemplary Tetrahymena permuted intron-exon with an annealing region of 6nucleotides (FIG. 9A) and an exemplary Tetrahymena permuted intron-exonwith an extended annealing region (FIG. 9B).

FIG. 11 is a graph showing circularization efficiency of Tetrahymenapermuted intron-exon with an annealing region of 6 nucleotides(Tetrahymena 1) and Tetrahymena permuted intron-exon with an extendedannealing region (Tetrahymena 2).

FIGS. 12A and 12B are schematic drawings showing an exemplary T4 phagepermuted intron-exon with an annealing region of 2 nucleotides (FIG.12A) and an exemplary T4 phage permuted intron-exon with an extendedannealing region (FIG. 12B).

FIG. 13 is a graph showing circularization efficiency of T4 phagepermuted intron-exon with an annealing region of 2 nucleotides (T4phage 1) and T4 phage permuted intron-exon with an extended annealingregion (T4 phage 2).

FIGS. 14A and 14B are schematic drawings showing an exemplary permutedintron-exon with an annealing region (FIG. 14A) and an exemplarypermuted intron-exon with an extended annealing region (FIG. 14B).

FIGS. 15A and 15B are schematic drawings showing the structures of anexemplary Synechococcus permuted intron-exon with an annealing region of7 nucleotides (FIG. 15A) and an exemplary Synechococcus permutedintron-exon with a modified and extended annealing region (FIG. 15B).

FIGS. 16A and 16B are schematic drawings showing the structures of anexemplary Anabaena azollae permuted intron-exon with an annealing regionof 5 nucleotides (FIG. 16A) and an exemplary Anabaena azollae permutedintron-exon with a modified and extended annealing region (FIG. 16B).

FIGS. 17A and 17B are schematic drawings showing the structures of anexemplary Anabaena cylindrica with an annealing region of 5 nucleotides(FIG. 17A) and an exemplary Anabaena cylindrica permuted intron-exonwith a modified and extended annealing region (FIG. 17B).

FIGS. 18A and 18B are schematic drawings showing the structures of anexemplary Scytonema permuted intron-exon with an annealing region of 5nucleotides (FIG. 18A) and an exemplary Scytonema permuted intron-exonwith a modified and extended annealing region (FIG. 18B).

FIG. 19 is a table showing exemplary modifications for various permutedintron-exon with an annealing region. Bolding identifies the originalannealing region; italics and underlining identify exemplarymodifications for extended annealing.

FIG. 20 is a graph showing fold increase of circularization of variousmodified permuted intron-exon with a 4.5 Kb RNA relative to theunmodified (original) permuted intron-exon with a 4.5 Kb RNA. Enhancedcircularization efficiency is observed with group I introns withpermuted intron-exon with extended E2-E1 annealing region.

FIG. 21A is a schematic drawing showing secondary structure of Anabaenaself-splicing intron. Permuting region in P6b is highlighted. FIG. 21Bare schematic drawings showing the structures of exemplary designs ofAnabaena permuted intron-exon with an extended P6b stem (Anabaena 4) orwith change of bulge of P6b to stem (Anabaena 5).

FIG. 22 is a graph showing circularization efficiency with the Anabaenapermuted intron-exon with an extended annealing region (Anabaena 2),Anabaena permuted intron-exon with an annealing region of 5 nucleotides(Anabaena 1), Anabaena 4, and Anabaena 5.

DETAILED DESCRIPTION

The present invention features compositions and methods for producing acircular polyribonucleotide (circular RNA). Circular polyribonucleotidesdescribed herein are particularly useful for delivering a polynucleotidecargo (e.g., encoding a gene or protein) to a target cell.

A circular polyribonucleotide may be produced from a linearpolyribonucleotide in which the ends are self-spliced together, therebyforming the circular polyribonucleotide. The linear RNA moleculesdescribed herein include, from 5′ to 3′, (A) a 3′ half of Group Icatalytic intron fragment; (B) a 3′ splice site; (C) a 3′ exon fragment;(D) a polyribonucleotide cargo; (E) a 5′ exon fragment; (F) a 5′ splicesite; and (G) a 5′ half of Group I catalytic intron fragment. Thepolyribonucleotide includes a first annealing region that has from 2 to50, e.g., from 8 to 50 ribonucleotides and is present within (A) the 3′half of Group I catalytic intron fragment; (B) the 3′ splice site; or(C) the 3′ exon fragment. The polyribonucleotide also includes a secondannealing region that has from 2 to 50, e.g., from 8 to 50ribonucleotides and is present within (E) the 5′ exon fragment; (F) the5′ splice site; or (G) the 5′ half of Group I catalytic intron fragment.The first annealing region has from 80% to 100% complementarity with thesecond annealing region or has from zero to 10 mismatched base pairs.These features allow the first annealing region to hybridize to thesecond annealing region, thus bringing the splice sites near the 5′ and3′ ends of the linear polyribonucleotide into close proximity. Once thesplice sites are nearby, the polyribonucleotide is able to self-splicethe 3′ and 5′ splice sites, thus forming the circularpolyribonucleotide.

By including the first annealing region within, for example, (A) the 3′half of Group I catalytic intron fragment; (B) the 3′ splice site; or(C) the 3′ exon fragment, and the second annealing region within, forexample, (E) the 5′ exon fragment; (F) the 5′ splice site; or (G) the 5′half of Group I catalytic intron fragment, the linear molecule exhibitsincreased circularization efficiency and splicing fidelity as comparedto other polyribonucleotide constructs that lack these features.Furthermore, by using an autocatalytic self-splicing intron, the linearmolecule does not need to be treated with an exogenous enzyme, such as aligase, to produce the circular polyribonucleotide. This is particularlyadvantageous for producing a circular product in a single pot reaction.The molecules, methods of producing, and uses thereof are described inmore detail below.

Polynucleotides

The disclosure features circular polyribonucleotide compositions andmethods of making circular polyribonucleotides. In some embodiments, acircular polyribonucleotide is produced from a linear polyribonucleotide(e.g., by self-splicing compatible ends of the linearpolyribonucleotide). In some embodiments, a linear polyribonucleotide istranscribed from a deoxyribonucleotide template (e.g., a vector, alinearized vector, or a cDNA). Accordingly, the disclosure featuresdeoxyribonucleotides, linear polyribonucleotides, and circularpolyribonucleotides and compositions thereof useful in the production ofcircular polyribonucleotides.

Template Deoxyribonucleotides

The present invention features a template deoxyribonucleotide for makingcircular RNA. The deoxyribonucleotide includes the following, operablylinked in a 5′-to-3′ orientation: (A) a 3′ half of Group I catalyticintron fragment; (B) a 3′ splice site; (C) a 3′ exon fragment; (D) apolyribonucleotide cargo; (E) a 5′ exon fragment; (F) a 5′ splice site;and (G) a 5′ half of Group I catalytic intron fragment. In embodiments,the deoxyribonucleotide includes further elements, e.g., outside of orbetween any of elements (A), (B), (C), (D), (E), (F), or (G). Inembodiments, any of the elements (A), (B), (C), (D), (E), (F), or (G) isseparated from each other by a spacer sequence, as described herein.

In embodiments, the deoxyribonucleotide is, for example, a circular DNAvector, a linearized DNA vector, or a linear DNA (e.g., a cDNA, e.g.,produced from a DNA vector).

In some embodiments, the deoxyribonucleotide further includes an RNApolymerase promoter operably linked to a sequence encoding a linear RNAdescribed herein. In embodiments, the RNA polymerase promoter isheterologous to the sequence encoding the linear RNA. In someembodiments, the RNA polymerase promoter is a T7 promoter, a T6promoter, a T4 promoter, a T3 promoter, an SP6 virus promoter, or an SP3promoter.

In some embodiments, the deoxyribonucleotide includes a multiple-cloningsite (MCS).

In some embodiments, the deoxyribonucleotide is used to produce circularRNA with the size range of about 100 to about 20,000 nucleotides. Insome embodiments, the circular RNA is at least 100, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or5,000 nucleotides in size. In some embodiments, the circular RNA is nomore than 20,000, 15,000 10,000, 9,000, 8,000, 7,000, 6,000, 5,000 or4,000 nucleotides in size.

Linear Polyribonucleotides

The present invention also features linear polyribonucleotides includingthe following, operably linked in a 5′-to-3′ orientation: (A) a 3′ halfof Group I catalytic intron fragment; (B) a 3′ splice site; (C) a 3′exon fragment; (D) a polyribonucleotide cargo; (E) a 5′ exon fragment;(F) a 5′ splice site; and (G) a 5′ half of Group I catalytic intronfragment. In embodiments, the linear polyribonucleotide includes furtherelements, e.g., outside of or between any of elements (A), (B), (C),(D), (E), (F), or (G). For example, any of elements (A), (B), (C), (D),(E), (F), or (G) may be separated by a spacer sequence, as describedherein.

In certain embodiments, provided herein is a method of generating linearRNA by performing transcription in a cell-free system (e.g., in vitrotranscription) using a deoxyribonucleotide (e.g., a vector, linearizedvector, or cDNA) provided herein as a template (e.g., a vector,linearized vector, or cDNA provided herein with an RNA polymerasepromoter positioned upstream of the region that codes for the linearRNA).

In embodiments, a deoxyribonucleotide template is transcribed to aproduce a linear RNA containing the components described herein. Uponexpression, the linear polyribonucleotide produces a splicing-compatiblepolyribonucleotide, which may be self-spliced in order to produce acircular polyribonucleotide.

In some embodiments, the linear polyribonucleotide is from 50 to 20,000,100 to 20,000, 200 to 20,000, 300 to 20,000 (e.g., 50, 100, 200, 300,400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000,6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000,15,000, 16,000, 17,000, 18,000, 19,000, or 20,000) ribonucleotides inlength. In embodiments, the linear polyribonucleotide is , e.g., atleast 500, at least 1,000, at least 2,000, at least 3,000, at least4,000, or at least 5,000 ribonucleotides in length.

Circular Polyribonucleotides

In some embodiments, the invention features a circularpolyribonucleotide (e.g., a covalently closed circularpolyribonucleotide). In embodiments, the circular polyribonucleotideincludes a splice junction joining a 5′ exon fragment and a 3′ exonfragment. In embodiments, the 3′ exon fragment includes the firstannealing region having from 2 to 50, e.g., from 8 to 50 (e.g., from 10to 30, 10 to 20, or 10 to 15, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50)ribonucleotides, and the 5′ exon fragment includes the second annealingregion having from 2 to 50, e.g., from 8 to 50 (e.g., from 10 to 30, 10to 20, or 10 to 15, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) ribonucleotides.In embodiments, the first annealing region and the second annealingregion include from 80% to 100% (e.g., 80%, 85%, 90%, 95%, 97%, 99%, or100%) complementarity. In embodiments, the first annealing region andthe second annealing region include from zero to 10 (e.g., 0, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10) mismatched base pairs.

In embodiments, the circular polynucleotide further includes apolyribonucleotide cargo. In embodiments, the polyribonucleotide cargoincludes an expression (or coding) sequence, a non-coding sequence, or acombination of an expression (coding) sequence and a non-codingsequence. In embodiments, the polyribonucleotide cargo includes anexpression (coding) sequence encoding a polypeptide. In embodiments, thepolyribonucleotide includes an IRES operably linked to an expressionsequence encoding a polypeptide. In some embodiments, the IRES islocated upstream of the expression sequence. In some embodiments, theIRES is located downstream of the expression sequence. In someembodiments, the circular polyribonucleotide further includes a spacerregion between the IRES and the 3′ exon fragment or the 5′ exonfragment. The spacer region may be, e.g., at least 5 (e.g., at least 10,at least 15, at least 20) ribonucleotides in length ribonucleotides inlength. The spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500)ribonucleotides. In some embodiments, the spacer region includes a polyAsequence. In some embodiments, the spacer region includes a polyA-Csequence. In some embodiments, the spacer region includes a polyA-Gsequence. In some embodiments, the spacer region includes a polyA-Tsequence. In some embodiments, the spacer region includes a randomsequence. In some embodiments, the first annealing region and the secondannealing region are joined, thereby forming a circularpolyribonucleotide.

In some embodiments, the circular RNA is a produced by adeoxyribonucleotide template or a linear RNA described herein. In someembodiments, the circular RNA is produced by any of the methodsdescribed herein.

In some embodiments, the circular polyribonucleotide is at least about20 nucleotides, at least about 30 nucleotides, at least about 40nucleotides, at least about 50 nucleotides, at least about 75nucleotides, at least about 100 nucleotides, at least about 200nucleotides, at least about 300 nucleotides, at least about 400nucleotides, at least about 500 nucleotides, at least about 1,000nucleotides, at least about 2,000 nucleotides, at least about 5,000nucleotides, at least about 6,000 nucleotides, at least about 7,000nucleotides, at least about 8,000 nucleotides, at least about 9,000nucleotides, at least about 10,000 nucleotides, at least about 12,000nucleotides, at least about 14,000 nucleotides, at least about 15,000nucleotides, at least about 16,000 nucleotides, at least about 17,000nucleotides, at least about 18,000 nucleotides, at least about 19,000nucleotides, or at least about 20,000 nucleotides.

In some embodiments, the circular polyribonucleotide is of a sufficientsize to accommodate a binding site for a ribosome. In some embodiments,the size of a circular polyribonucleotide is a length sufficient toencode useful polypeptides, e.g., at least 20,000 nucleotides, at least15,000 nucleotides, at least 10,000 nucleotides, at least 7,500nucleotides, at least 5,000 nucleotides, at least 4,000 nucleotides, atleast 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000nucleotides, at least 500 nucleotides, at least 1400 nucleotides, atleast 300 nucleotides, at least 200 nucleotides, or at least 100nucleotides may be produced.

In some embodiments, the circular polyribonucleotide includes one ormore elements described elsewhere herein. In some embodiments, theelements are separated from one another by a spacer sequence. In someembodiments, the elements are separated from one another by 1ribonucleotide, 2 nucleotides, about 5 nucleotides, about 10nucleotides, about 15 nucleotides, about 20 nucleotides, about 30nucleotides, about 40 nucleotides, about 50 nucleotides, about 60nucleotides, about 80 nucleotides, about 100 nucleotides, about 150nucleotides, about 200 nucleotides, about 250 nucleotides, about 300nucleotides, about 400 nucleotides, about 500 nucleotides, about 600nucleotides, about 700 nucleotides, about 800 nucleotides, about 900nucleotides, about 1000 nucleotides, up to about 1 kb, at least about1000 nucleotides, or any amount of nucleotides therebetween. In someembodiments, one or more elements are contiguous with one another, e.g.,lacking a spacer element.

In some embodiments, the circular polyribonucleotide includes one ormore repetitive elements described elsewhere herein. In someembodiments, the circular polyribonucleotide includes one or moremodifications described elsewhere herein. In one embodiment, thecircular RNA contains at least one nucleoside modification. In oneembodiment, up to 100% of the nucleosides of the circular RNA aremodified. In one embodiment, at least one nucleoside modification is auridine modification or an adenosine modification.

As a result of its circularization, the circular polyribonucleotide mayinclude certain characteristics that distinguish it from linear RNA. Forexample, the circular polyribonucleotide is less susceptible todegradation by exonuclease as compared to linear RNA. As such, thecircular polyribonucleotide is more stable than a linear RNA, especiallywhen incubated in the presence of an exonuclease. The increasedstability of the circular polyribonucleotide compared with linear RNAmakes circular polyribonucleotide more useful as a cell transformingreagent to produce polypeptides and can be stored more easily and forlonger than linear RNA. The stability of the circular polyribonucleotidetreated with exonuclease can be tested using methods standard in artwhich determine whether RNA degradation has occurred (e.g., by gelelectrophoresis). Moreover, unlike linear RNA, the circularpolyribonucleotide is less susceptible to dephosphorylation when thecircular polyribonucleotide is incubated with phosphatase, such as calfintestine phosphatase.

Annealing Regions

Polynucleotide compositions described herein may include two or moreannealing regions, e.g., two or more annealing regions described herein.An annealing region, or pair of annealing regions, are those thatcontain a portion with a high degree of complementarity that promoteshybridization under suitable conditions.

An annealing region includes at least a region of complementary asdescribed herein. The high degree of complementarity of thecomplementary region promotes the association of annealing region pairs.When a first annealing region (e.g., a 5′ annealing region) is locatedat or near the 5′ end of a linear RNA and a second annealing region(e.g., a 3′ annealing region) is located at or near the 3′ end of alinear RNA, association of the annealing regions brings the 5′ and 3′and the corresponding intron fragments into proximity. In someembodiments, this favor circularization of the linear RNA by splicing ofthe 3′ and 5′ splice sites. In some embodiments, the annealing regionsdescribed herein strengthen naturally occurring annealing regions, e.g.,to promote self-splicing.

An annealing region may be altered by introducing one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore) mutations into the polyribonucleotide sequence. For example, anannealing region may be extended by introducing one or more pointmutations into a first annealing region and/or a second annealing regionto increase the length of complementarity between the first and secondannealing regions. The annealing region may also be altered by insertingone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more) nucleotides into the polyribonucleotide. Inembodiments, an annealing region is extended by inserting one or morenucleotides into a first annealing region and/or a second annealingregion to increase the length of complementarity between the first andsecond annealing regions. In embodiments, the annealing region isextended by introducing one or more point mutations into a firstannealing and/or a second region and inserting one or more nucleotidesinto the first annealing and/or the second annealing region to increasethe length of complementarity. Altering the annealing region may alterthe secondary structure of the polyribonucleotide by favoring a bulge ormismatched region with the original sequence to preferentially form astem or stem loop structure with the altered sequence.

The polyribonucleotide includes a first annealing region that has from 2to 50, 5 to 50, 6 to 50, 7 to 50, or 8 to 50 (e.g., from 10 to 30, 10 to20, or 10 to 15, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50)ribonucleotides and is present within (A) the 3′ half of Group Icatalytic intron fragment; (B) the 3′ splice site; or (C) the 3′ exonfragment. The polyribonucleotide also includes a second annealing regionthat has from 2 to 50, 5 to 50, 6 to 50, 7 to 50, or 8 to 50 (e.g., from10 to 30, 10 to 20, or 10 to 15, e.g., at least 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50) ribonucleotides and is present within (E) the 5′ exonfragment; (F) the 5′ splice site; or (G) the 5′ half of Group Icatalytic intron fragment. The first annealing region has from 80% to100% (e.g., 85% to 100%, e.g., 90% to 100%, e.g., 80%, 85%, 90%, 95%,97%, 99%, or 100%) complementarity with the second annealing region orhas from zero to 10 e.g., (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)mismatched base pairs.

In some embodiments, the first annealing region and the second annealingregion are 100% complementary.

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- TCCGT-3′ (SEQ ID NO: 1), and the second annealing regionhas at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of 5′- ACGGA-3′ (SEQ ID NO: 2).

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- TCCGTAGCGTCT -3′ (SEQ ID NO: 5), and the secondannealing region has at least 80% (e.g., at least 85%, 90%, 95%, 97%,99%, or 100%) sequence identity to the sequence of 5′- AGACGCTACGGA -3′(SEQ ID NO: 6).

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- TCCGTAGCGTCTAAACG -3′ (SEQ ID NO: 22), and the secondannealing region has at least 80% (e.g., at least 85%, 90%, 95%, 97%,99%, or 100%) sequence identity to the sequence of 5′- CGTTTAGACGCTACGGA-3′ (SEQ ID NO: 23).

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- TCCGTAGCGTCTAAACGGTCGT -3′ (SEQ ID NO: 24), and thesecond annealing region has at least 80% (e.g., at least 85%, 90%, 95%,97%, 99%, or 100%) sequence identity to the sequence of 5′-ACGACCGTTTAGACGCTACGGA -3′ (SEQ ID NO: 25).

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- TCCGTAGCGTCTAAACGGTCGTGTGGG -3′ (SEQ ID NO: 26), and thesecond annealing region has at least 80% (e.g., at least 85%, 90%, 95%,97%, 99%, or 100%) sequence identity to the sequence of 5′-CCCACACGACCGTTTAGACGCTACGGA -3′ (SEQ ID NO: 27).

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- AAGGTA -3′ (SEQ ID NO: 13), and the second annealingregion has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or100%) sequence identity to the sequence of 5′- TACCTT -3′ (SEQ ID NO:14).

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- AAGGTAAATATT -3′ (SEQ ID NO: 16), and the secondannealing region has at least 80% (e.g., at least 85%, 90%, 95%, 97%,99%, or 100%) sequence identity to the sequence of 5′- AATATTTACCTT -3′(SEQ ID NO: 17).

In some embodiments, the first annealing region has the sequence of 5′-CT -3′, and the second annealing region has the sequence of 5′- AG -3′.

In some embodiments, the first annealing region has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of 5′- CTCAATT -3′ (SEQ ID NO: 20), and the second annealingregion has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or100%) sequence identity to the sequence of 5′- AATTGAG -3′ (SEQ ID NO:21).

In some embodiments, (A) or (C) includes the first annealing region and(E) or (G) includes the second annealing region.

In some embodiments, the 3′ exon fragment of (C) includes the firstannealing region and the 5′ exon fragment of (E) includes the secondannealing region.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) includes the first annealing region and the 5′ exon fragment of (E)includes the second annealing region.

In some embodiments, the 3′ exon fragment of (C) includes the firstannealing region and the 5′ half of Group I catalytic intron fragmentincludes the second annealing region.

In some embodiments, first annealing region and the second annealingregion include zero or one mismatched base pair.

In embodiments, an annealing region further includes a non-complementaryregion as described below. A non-complementary region may be added tothe complementary region to allow for the ends of the RNA to remainflexible, unstructured, or less structured than the complementarityregion.

In some embodiments, each annealing region includes 2 to 100, 5 to 100,or 6 to 100 ribonucleotides (e.g., 6 to 80, 6 to 50, 6 to 30, 6 to 20,10 to 100, 10 to 80, 10 to 50, or 10 to 30 ribonucleotides). In someembodiments, a 5′ annealing region includes 2 to 100, 5 to 100, 6 to 100ribonucleotides (e.g., 6 to 80, 6 to 50, 6 to 30, 6 to 20, 10 to 100, 10to 80, 10 to 50, or 10 to 30 ribonucleotides). In some embodiments, a 3′annealing region includes 6 to 100 ribonucleotides (e.g., 6 to 80, 6 to50, 6 to 30, 6 to 20, 10 to 100, 10 to 80, 10 to 50, or 10 to 30ribonucleotides).

In some embodiments, the polyribonucleotide does not include anannealing region 3′ to (A) that includes partial or complete nucleicacid complementarity with an annealing region 5′ to (G).

In some embodiments, the polyribonucleotide does not include a furtherannealing region, e.g., in addition to the first annealing region andsecond annealing region.

Complementary Regions

A complementary region is a region that favors association with acorresponding complementary region, under suitable conditions. Forexample, a pair of complementary regions may share a high degree ofsequence complementarity (e.g., a first complementary region is thereverse complement of a second complementary region, at least in part).When two complementary regions associate (e.g., hybridize), they mayform a highly structured secondary structure, such as a stem or stemloop.

In some embodiments, the polyribonucleotide includes a 5′ complementaryregion and a 3′ complementary region. In some embodiments, the 5′complementary region has from 2 to 50, e.g., 5 to 50 ribonucleotides(e.g., 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50,e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ribonucleotides). In someembodiments, the 3′ complementary region has from 2 to 50, e.g., 5 to 50ribonucleotides (e.g., 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30,10-20, or 20-50, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50ribonucleotides).

In some embodiments, the 5′ complementary region and the 3′complementary region have from 50% to 100% sequence complementarity(e.g., from 60%-100%, 70%-100%, 80%-100%, 90%-100%, or 100%, e.g., 80%,85%, 90%, 95%, 97%, 99%, or 100% sequence complementarity).

In some embodiments, the 5′ complementary region and the 3′complementary region have a free energy of binding of less than -5kcal/mol (e.g., less than -10 kcal/mol, less than -20 kcal/mol, or lessthan -30 kcal/mol).

In some embodiments, the 5′ complementary region and the 3′complementary region have a Tm of binding of at least 10° C., at least15° C., at least 20° C., at least 30° C., at least 40° C., at least 50°C., at least 60° C., at least 70° C., at least 80° C., or at least 90°C.

In some embodiments, the 5′ complementary region and the 3′complementary region include at least one but no more than 10mismatches, e.g., 10, 9, 8, 7, 6, 5, 4, 3, or 2 mismatches, or 1mismatch (i.e., when the 5′ complementary region and the 3′complementary region hybridize to each other). A mismatch can be, e.g.,a nucleotide in the 5′ complementary region and a nucleotide in the 3′complementary region that are opposite each other (i.e., when the 5′complementary region and the 3′ complementary region are hybridized) butthat do not form a Watson-Crick base-pair. A mismatch can be, e.g., anunpaired nucleotide that forms a kink or bulge in either the 5′complementary region or the 3′ complementary region. In someembodiments, the 5′ complementary region and the 3′ complementary regiondo not include any mismatches.

Non-Complementary Regions

A non-complementary region is a region that disfavors association with acorresponding non-complementary region, under suitable conditions. Forexample, a pair of non-complementary regions may share a low degree ofsequence complementarity (e.g., a first non-complementary region is nota reverse complement of a second non-complementary region). When twonon-complementary regions are in proximity, they do not form a highlystructured secondary structure, such as a stem or stem loop.

In some embodiments, the polyribonucleotide includes a 5′non-complementary region and a 3′ non-complementary region. In someembodiments, the 5′ non-complementary region has from 5 to 50ribonucleotides (e.g., 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30,10-20, or 20-50 ribonucleotides). In some embodiments, the 3′non-complementary region has from 5 to 50 ribonucleotides (e.g., 5-40,5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50 ribonucleotides).

In some embodiments the 5′ non-complementary region is located 5′ to the5′ complementary region (e.g., between the 5′ catalytic intron fragmentand the 5′ complementary region). In some embodiments, the 3′non-complementary region is located 3′ to the 3′ complementary region(e.g., between the 3′ complementary region and the 3′ catalytic intronfragment).

In some embodiments, the 5′ non-complementary region and the 3′non-complementary region have from 0% to 50% sequence complementarity(e.g., from 0%-40%, 0%-30%, 0%-20%, 0%-10%, or 0% sequencecomplementarity).

In some embodiments, the 5′ non-complementary region and the 3′non-complementary region have a free energy of binding of greater than-5 kcal/mol.

In some embodiments, the 5′ complementary region and the 3′complementary region have a Tm of binding of less than 10° C.

In some embodiments, the 5′ non-complementary region and the 3′non-complementary region include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 mismatches.

Catalytic Introns

The polyribonucletides described herein include catalytic intronfragments, such as (A) a 3′ half of Group I catalytic intron fragmentand (G) a 5′ half of Group I catalytic intron fragment. The first andsecond annealing regions may be positioned within the catalytic intronfragments. Group I catalytic introns are self-splicing ribozymes thatcatalyze their own excision from mRNA, tRNA, and rRNA precursors viatwo-metal ion phorphoryl transfer mechanism. Importantly, the RNA itselfself-catalyzes the intron removal without the requirement of anexogenous enzyme, such as a ligase.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ half of Group I catalytic intron fragment of (G) are froma cyanobacterium Anabaena pre-tRNA-Leu gene, or a Tetrahymena pre-rRNA.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ half of Group I catalytic intron fragment of (G) are froma Cyanobacterium Anabaena pre-tRNA-Leu gene, and the 3′ exon fragment of(C) includes the first annealing region and the 5′ exon fragment of (E)includes the second annealing region. The first annealing region mayinclude, e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11, 12, 13,14, or 15) ribonucleotides and the second annealing region may include,e.g., from 5 to 50, e.g., from 10 to 15 (e.g., 10, 11, 12, 13, 14, or15) ribonucleotides.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ half of Group I catalytic intron fragment of (G) are froma Tetrahymena pre-rRNA, and the 3′ half of Group I catalytic intronfragment of (A) includes the first annealing region and the 5′ exonfragment of (E) includes the second annealing region. In someembodiments, the 3′ exon of (B) includes the first annealing region andthe 5′ half of Group I catalytic intron fragment of (G) includes thesecond annealing region. The first annealing region may include, e.g.,from 6 to 50, e.g., from 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16)ribonucleotides, and the second annealing region may include, e.g., from6 to 50, e.g., from 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16)ribonucleotides.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ half of Group I catalytic intron fragment of (G) are froma cyanobacterium Anabaena pre-tRNA-Leu gene, a Tetrahymena pre-rRNA, ora T4 phage td gene.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) and the 5′ Group I catalytic intron fragment of (G) are from a T4phage td gene. The 3′ exon fragment of (C) may include the firstannealing region and the 5′ half of Group I catalytic intron fragment of(G) may include the second annealing region. The first annealing regionmay include, e.g., from 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the secondannealing region may include, e.g., from 2 to 16, e.g., 10 to 16 (e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) is the 5′ terminus of the linear polynucleotide.

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) is the 3′ terminus of the linear polyribonucleotide.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AACAACAGATAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATG-3′ (SEQ ID NO:  28).

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT-3′ (SEQ ID NO: 29).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 28 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 29.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-CTTCTGTTGATATGGATGCAGTTCACAGACTAAATGTCGGTCGGGGAAGATGTATTCTTCTCATAAGATATAGTCGGACCTCTCCTTAATGGGAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTATGCGAAAGTATATTGATTAGTTTTGGAGTACTCG-3′ (SEQ ID NO: 30) .

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AAATAGCAATATTTACCTTTGGAGGGAAAAGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAAAAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGGGGAAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAACCACGCAGCCAAGTCCTAAGTCAACAGAT-3′ (SEQ ID  NO: 31).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 30 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 31.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-GGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATG-3′ (SEQ ID NO: 32).

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACT-3′  (SEQ ID NO: 33).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 32 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 33.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-TAAACAACTAACAGCTTTAGAAGGTGCAGAGACTAGACGGGAGCTACCCTAACGGATTCAGCCGAGGGTAAAGGGATAGTCCAATTCTCAACATCGCGATTGTTGATGGCAGCGAAAGTTGCAGAGAGAATGAAAATCCGCTGACTGTAAAGGTCGTGAGGGTTCGAGTCCCTCCGCCCCCA-3′ (SEQ ID NO:  80).

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-ACGGTAGACGCAGCGGACTTAGAAAACTGGGCCTCGATCGCGAAAGGGATCGAGTGGCAGCTCTCAAACTCAGGGAAACCTAAAACTTTAAACATTMAAGTCATGGCAATCCTGAGCCAAGCTAAAGC-3′ (SEQID NO: 81).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 80 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 81.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-TTAAACTCAAAATTTAAAATCCCAAATTCAAAATTCCGGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTAAAGCCGAGGGTAAAGGGAGAGTCCAATTCTCAAAGCCTGAAGTTGCTGAAGCAACAAGGCAGTAGTGAAAGCTGCGAGAGAATGAAAATCCGTTGACTGTAAAAAGTCGTGGGGGTTCAAGTCCCCCCACCCCC-3′ (SEQ ID NO: 82).

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTTAAGTGGAAGCTCTCAAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCAGAAAATTTAGACTTGAGATTTGATTTTGGAG-3′ (SEQ ID NO: 83).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 82 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 83.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-GGCTTTCAATTTGAAATCAGAAATTCAAAATTCAGGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTAAAGGCGAGGGTAAAGGGAGAGTCCAATTCTTAAAGCCTGAAGTTGTGCAAGCAACAAGGCAACAGTGAAAGCTGTGGAAGAATGAAAATCCGTTGACCTTAAACGGTCGTGGGGGTTCAAGTCCCCCCACCCCC-3′ (SEQ ID NO: 84).

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTCAAGTGGAAGCTCTCAAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCGGAAATTTTAGAATCAAGATTTTATTTT -3′ (SEQ ID NO: 85).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 84 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 85.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGGGACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCGAAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA-3′ (SEQ ID NO: 86) .

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGGGACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCGAAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA-3′ (SEQ ID NO: 87) .

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 86 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 87.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-ACAACAGATAACTTACTAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3′ (SEQ  ID NO:88).

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAGTAAGTT-3′ (S EQ ID NO: 89).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 88 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 89.

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AACAACAGATAACTTACTAGTTACTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3′ (SEQ ID  NO: 90).

In some embodiments, the 5′ half of Group I catalytic intron fragment of(G) has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%)sequence identity to the sequence of

5′-AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT-3′(SEQ ID NO:  91).

In some embodiments, the 3′ half of Group I catalytic intron fragment of(A) has the sequence of SEQ ID NO: 90 and the 5′ half of Group Icatalytic intron fragment of (G) has the sequence of SEQ ID NO: 91.

Splice Sites

The polyribonucleotides described herein include splice sites, such as(B) a 3′ splice site; and (F) a 5′ splice site. The splice site may befrom a cyanobacterium Anabaena pre-tRNA-Leu gene, a Tetrahymenapre-rRNA, or a T4 phage td gene.

In some embodiments the 3′ splice site (e.g., between the 3′ half ofGroup I catalytic intron fragment and the 3′ exon fragment has thesequence of AGAATG ↓ AAAATC (SEQ ID NO: 34) where the arrow denotes thecut site. In some embodiments, the 5′ splice site (e.g., between the 5′exon fragment and the 5′ half of Group I catalytic intron fragment hasthe sequence of GGACTT ↓ AAATAA (SEQ ID NO: 35) where the arrow denotesthe cut site.

In some embodiments the 3′ splice site (e.g., between the 3′ half ofGroup I catalytic intron fragment and the 3′ exon fragment has thesequence TACTCG I TAAGGT (SEQ ID NO: 36) where the arrow denotes the cutsite. In some embodiments, the 5′ splice site (e.g., between the 5′ exonfragment and the 5′ half of Group I catalytic intron fragment has thesequence of CTCTCT ↓ AAATAG (SEQ ID NO: 37) where the arrow denotes thecut site.

In some embodiments the 3′ splice site (e.g., between the 3′ half ofGroup I catalytic intron fragment and the 3′ exon fragment has thesequence of ATAATG I CTACCG (SEQ ID NO: 38) where the arrow denotes thecut site. In some embodiments, the 5′ splice site (e.g., between the 5′exon fragment and the 5′ half of Group I catalytic intron fragment hasthe sequence of TTGGGT ↓ TAATTG (SEQ ID NO: 39) where the arrow denotesthe cut site.

Exon Fragments

The polyribonucleotides described herein include an exon fragment, suchas (C) a 3′ exon fragment; and (E) a 5′ exon fragment.

In some embodiments, the 3′ exon fragment of (C) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-AAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3′ (SEQ ID NO: 40).

In some embodiments, the 3′ exon fragment of (C) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-AAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA-3′ (SEQ ID NO: 41).

In some embodiments, the 5′ exon fragment of (E) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′- AGACGCTACGGACTT-3′ (SEQ ID NO: 42).

In some embodiments, the 5′ exon fragment of (E) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-CGTTTAGACGCTACGGACTT-3′ (SEQ ID NO: 43).

In some embodiments, the 5′ exon fragment of (E) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-ACGACCGTTTAGACGCTACGGACTT-3′ (SEQ ID NO: 44).

In some embodiments, the 5′ exon fragment of (E) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-CCCACACGACCGTTTAGACGCTACGGACTT-3′ (SEQ ID NO: 45).

In some embodiments, the 3′ exon fragment of (C) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′- TAAGGTAGC-3′ (SEQ ID NO: 46).

In some embodiments, the 3′ exon fragment of (C) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-TAAGGTAAATATTGC-3′ (SEQ ID NO: 47).

In some embodiments, the 5′ exon fragment of (E) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-ATGACTCTCT-3′ (SEQ ID NO: 48).

In some embodiments, the 3′ exon fragment of (C) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-CTACCGTTTAATATT-3′ (SEQ ID NO: 49).

In some embodiments, the 3′ exon fragment of (C) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-CTCAATTTTAATATT-3′ (SEQ ID NO: 50).

In some embodiments, the 5′ exon fragment of (E) has at least 80% (e.g.,at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to thesequence of

5′-ATGTTTTCTTGGGT-3′ (SEQ ID NO: 51).

Polyribonucleotide Cargo

A polyribonucleotide cargo described herein includes any sequenceincluding at least one polyribonucleotide. In some embodiments, thepolyribonucleotide cargo of (D) includes an expression sequence, anon-coding sequence, or an expression sequence and a non-codingsequence. In some embodiments, the polyribonucleotide cargo of (D)includes an expression sequence encoding a polypeptide. In someembodiments, the polyribonucleotide cargo of (D) includes an IRESoperably linked to an expression sequence encoding a polypeptide. Insome embodiments, the polyribonucleotide cargo of (D) includes anexpression sequence that encodes a polypeptide that has a biologicaleffect on a subject.

A polyribonucleotide cargo may, for example, include at least about 40nucleotides, at least about 50 nucleotides, at least about 75nucleotides, at least about 100 nucleotides, at least about 200nucleotides, at least about 300 nucleotides, at least about 400nucleotides, at least about 500 nucleotides, at least about 1,000nucleotides, at least about 2,000 nucleotides, at least about 5,000nucleotides, at least about 6,000 nucleotides, at least about 7,000nucleotides, at least about 8,000 nucleotides, at least about 9,000nucleotides, at least about 10,000 nucleotides, at least about 12,000nucleotides, at least about 14,000 nucleotides, at least about 15,000nucleotides, at least about 16,000 nucleotides, at least about 17,000nucleotides, at least about 18,000 nucleotides, at least about 19,000nucleotides, or at least about 20,000 nucleotides. In some embodiments,the polyribonucleotides cargo includes from 1-20,000 nucleotides,1-10,000 nucleotides, 1-5,000 nucleotides, 100-20,000 nucleotide,100-10,000 nucleotides, 100-5,000 nucleotides, 500-20,000 nucleotides,500-10,000 nucleotides, 500-5,000 nucleotides, 1,000-20,000 nucleotides,1,000-10,000 nucleotides, or 1,000-5,000 nucleotides.

In embodiments, the polyribonucleotide cargo includes one or multipleexpression (or coding) sequences, wherein each expression (or coding)sequence encodes a polypeptide. In embodiments, the polyribonucleotidecargo includes one or multiple noncoding sequences. In embodiments, thepolyribonucleotide cargo consists entirely of non-coding sequence(s). Inembodiments, the polyribonucleotide cargo includes a combination ofexpression (or coding) and noncoding sequences.

In some embodiments, polyribonucleotides made as described herein areused as effectors in therapy or agriculture. For example, a circularpolyribonucleotide made by the methods described herein (e.g., thecell-free methods described herein) may be administered to a subject(e.g., in a pharmaceutical, veterinary, or agricultural composition). Inanother example, a circular polyribonucleotide made by the methodsdescribed herein (e.g., the cell-free methods described herein) may bedelivered to a cell.

In some embodiments, the polyribonucleotide includes any feature, or anycombination of features as disclosed in International Patent PublicationNo. WO2019/118919, which is hereby incorporated by reference in itsentirety.

Polypeptide Expression Sequences

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the circular polyribonucleotide) includesone or more expression (or coding) sequences, wherein each expressionsequence encodes a polypeptide. In some embodiments, the circularpolyribonucleotide includes two, three, four, five, six, seven, eight,nine, ten or more expression (or coding) sequences.

Each encoded polypeptide may be linear or branched. In variousembodiments, the polypeptide has a length from about 5 to about 40,000amino acids, about 15 to about 35,000 amino acids, about 20 to about30,000 amino acids, about 25 to about 25,000 amino acids, about 50 toabout 20,000 amino acids, about 100 to about 15,000 amino acids, about200 to about 10,000 amino acids, about 500 to about 5,000 amino acids,about 1,000 to about 2,500 amino acids, or any range therebetween. Insome embodiments, the polypeptide has a length of less than about 40,000amino acids, less than about 35,000 amino acids, less than about 30,000amino acids, less than about 25,000 amino acids, less than about 20,000amino acids, less than about 15,000 amino acids, less than about 10,000amino acids, less than about 9,000 amino acids, less than about 8,000amino acids, less than about 7,000 amino acids, less than about 6,000amino acids, less than about 5,000 amino acids, less than about 4,000amino acids, less than about 3,000 amino acids, less than about 2,500amino acids, less than about 2,000 amino acids, less than about 1,500amino acids, less than about 1,000 amino acids, less than about 900amino acids, less than about 800 amino acids, less than about 700 aminoacids, less than about 600 amino acids, less than about 500 amino acids,less than about 400 amino acids, less than about 300 amino acids, orless may be useful.

Polypeptides included herein may include naturally occurringpolypeptides or non-naturally occurring polypeptides. In someembodiments, the polypeptide is or includes a functional fragment orvariant of a reference polypeptide (e.g., an enzymatically activefragment or variant of an enzyme). For example, the polypeptide may be afunctionally active variant of any of the polypeptides described hereinwith at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region orover the entire sequence, to a sequence of a polypeptide describedherein or a naturally occurring polypeptide. In some instances, thepolypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%,90%, 95%, 97%, 99%, or greater) identity to a protein of interest.

Some examples of a polypeptide include, but are not limited to, afluorescent tag or marker, an antigen, a therapeutic polypeptide, or apolypeptide for agricultural applications.

A therapeutic polypeptide may be a hormone, a neurotransmitter, a growthfactor, an enzyme (e.g., oxidoreductase, metabolic enzyme, mitochondrialenzyme, oxygenase, dehydrogenase, ATP -independent enzyme, lysosomalenzyme, desaturase), a cytokine, an antigen binding polypeptide (e.g.,antigen binding antibody or antibody-like fragments, such as singlechain antibodies, nanobodies or other Ig heavy chain or light chaincontaining polypeptides), an Fc fusion protein, an anticoagulant, ablood factor, a bone morphogenetic protein, an interferon, aninterleukin, and a thrombolytic.

A polypeptide for agricultural applications may be a bacteriocin, alysin, an antimicrobial polypeptide, an antifungal polypeptide, a noduleC-rich peptide, a bacteriocyte regulatory peptide, a peptide toxin, apesticidal polypeptide (e.g., insecticidal polypeptide or nematocidalpolypeptide), an antigen binding polypeptide (e.g., antigen bindingantibody or antibody-like fragments, such as single chain antibodies,nanobodies or other Ig heavy chain or light chain containingpolypeptides), an enzyme (e.g., nuclease, amylase, cellulase, peptidase,lipase, chitinase), a peptide pheromone, and a transcription factor.

In some cases, the circular polyribonucleotide expresses a non-humanprotein.

In some embodiments, the circular polyribonucleotide expresses anantibody, e.g., an antibody fragment, or a portion thereof. In someembodiments, the antibody expressed by the circular polyribonucleotidecan be of any isotype, such as IgA, IgD, IgE, IgG, IgM. In someembodiments, the circular polyribonucleotide expresses a portion of anantibody, such as a light chain, a heavy chain, a Fc fragment, a CDR(complementary determining region), a Fv fragment, or a Fab fragment, afurther portion thereof. In some embodiments, the circularpolyribonucleotide expresses one or more portions of an antibody. Forinstance, the circular polyribonucleotide can include more than oneexpression (or coding) sequence, each of which expresses a portion of anantibody, and the sum of which can constitute the antibody. In somecases, the circular polyribonucleotide includes one expression sequencecoding for the heavy chain of an antibody, and another expressionsequence coding for the light chain of the antibody. In some cases, whenthe circular polyribonucleotide is expressed in a cell or a cell-freeenvironment, the light chain and heavy chain can be subject toappropriate modification, folding, or other post-translationmodification to form a functional antibody.

In embodiments, polypeptides include multiple polypeptides, e.g.,multiple copies of one polypeptide sequence, or multiple differentpolypeptide sequences. In embodiments, multiple polypeptides areconnected by linker amino acids or spacer amino acids.

In embodiments, the polynucleotide cargo includes a sequence encoding asignal peptide. Many signal peptide sequences have been described, forexample, the Tat (Twin-arginine translocation) signal sequence istypically an N-terminal peptide sequence containing a consensus SRRxFLK“twin-arginine” motif, which serves to translocate a folded proteincontaining such a Tat signal peptide across a lipid bilayer. See also,e.g., the Signal Peptide Database publicly available atwww[dot]signalpeptide[dot]de. Signal peptides are also useful fordirecting a protein to specific organelles; see, e.g., theexperimentally determined and computationally predicted signal peptidesdisclosed in the Spdb signal peptide database, publicly available atproline[dot]bic[dot]nus[dot]edu[dot]sg/spdb.

In embodiments, the polynucleotide cargo includes sequence encoding acell-penetrating peptide (CPP). Hundreds of CPP sequences have beendescribed; see, e.g., the database of cell-penetrating peptides,CPPsite, publicly available at crdd[dot]osdd[dot]net/raghava/cppsite/.An example of a commonly used CPP sequence is a poly-arginine sequence,e.g., octoarginine or nonoarginine, which can be fused to the C-terminusof the CGI peptide.

In embodiments, the polynucleotide cargo includes sequence encoding aself-assembling peptide; see, e.g., Miki et al. (2021) NatureCommunications, 21 :3412, DOI: 10.1038/s41467-021-23794-6.

In some embodiments, the expression (or coding) sequence includes apoly-A sequence (e.g., at the 3′ end of an expression sequence). In someembodiments, the length of a poly-A sequence is greater than 10nucleotides in length. In one embodiment, the poly-A sequence is greaterthan 15 nucleotides in length (e.g., at least or greater than about 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160,180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000,1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,2,500, and 3,000 nucleotides). In some embodiments, the poly-A sequenceis designed according to the descriptions of the poly-A sequence in[0202]-[0204] of International Patent Publication No. WO2019/118919A1,which is incorporated herein by reference in its entirety. In someembodiments, the expression sequence lacks a poly-A sequence (e.g., atthe 3′ end of an expression sequence).

In some embodiments, a circular polyribonucleotide includes a polyA,lacks a polyA, or has a modified polyA to modulate one or morecharacteristics of the circular polyribonucleotide. In some embodiments,the circular polyribonucleotide lacking a polyA or having modified polyAimproves one or more functional characteristics, e.g., immunogenicity(e.g., the level of one or more marker of an immune or inflammatoryresponse), half-life, and/or expression efficiency.

Therapeutic Polypeptides

In some embodiments, the circular polyribonucleotide described herein(e.g., the polyribonucleotide cargo of the circular polyribonucleotide)includes at least one expression sequence encoding a therapeuticpolypeptide. A therapeutic polypeptide is a polypeptide that whenadministered to or expressed in a subject provides some therapeuticbenefit. Administration to a subject or expression in a subject of atherapeutic polypeptide may be used to treat or prevent a disease,disorder, or condition or a symptom thereof. In some embodiments, thecircular polyribonucleotide encodes two, three, four, five, six, seven,eight, nine, ten or more therapeutic polypeptides.

In some embodiments, the circular polyribonucleotide includes anexpression sequence encoding a therapeutic protein. The protein maytreat the disease in the subject in need thereof. In some embodiments,the therapeutic protein can compensate for a mutated, under-expressed,or absent protein in the subject in need thereof. In some embodiments,the therapeutic protein can target, interact with, or bind to a cell,tissue, or virus in the subject in need thereof.

A therapeutic polypeptide can be a polypeptide that can be secreted froma cell, or localized to the cytoplasm, nucleus, or membrane compartmentof a cell.

A therapeutic polypeptide may be a hormone, a neurotransmitter, a growthfactor, an enzyme (e.g., oxidoreductase, metabolic enzyme, mitochondrialenzyme, oxygenase, dehydrogenase, ATP -independent enzyme, lysosomalenzyme, desaturase), a cytokine, a transcription factor, an antigenbinding polypeptide (e.g., antigen binding antibody or antibody-likefragments, such as single chain antibodies, nanobodies or other Ig heavychain or light chain containing polypeptides), an Fc fusion protein, ananticoagulant, a blood factor, a bone morphogenetic protein, aninterferon, an interleukin, a thrombolytic, an antigen (e.g., a tumor,viral, or bacterial antigen), a nuclease (e.g., an endonuclease such asa Cas protein, e.g., Cas9), a membrane protein (e.g., a chimeric antigenreceptor (CAR), a transmembrane receptor, a G-protein-coupled receptor(GPCR), a receptor tyrosine kinase (RTK), an antigen receptor, an ionchannel, or a membrane transporter), a secreted protein, a gene editingprotein (e.g., a CRISPR-Cas, TALEN, or zinc finger), or a gene writingprotein (see, e.g., International Patent Publication No. WO2020/047124,incorporated in its entirety herein by reference).

In some embodiments, the therapeutic polypeptide is an antibody, e.g., afull-length antibody, an antibody fragment, or a portion thereof. Insome embodiments, the antibody expressed by the circularpolyribonucleotide can be of any isotype, such as IgA, IgD, IgE, IgG,IgM. In some embodiments, the circular polyribonucleotide expresses aportion of an antibody, such as a light chain, a heavy chain, a Fcfragment, a CDR (complementary determining region), a Fv fragment, or aFab fragment, a further portion thereof. In some embodiments, thecircular polyribonucleotide expresses one or more portions of anantibody. For instance, the circular polyribonucleotide can include morethan one expression sequence, each of which expresses a portion of anantibody, and the sum of which can constitute the antibody. In somecases, the circular polyribonucleotide includes one expression sequencecoding for the heavy chain of an antibody, and another expressionsequence coding for the light chain of the antibody. When the circularpolyribonucleotide is expressed in a cell, the light chain and heavychain can be subject to appropriate modification, folding, or otherpost-translation modification to form a functional antibody.

In some embodiments, circular polyribonucleotides made as describedherein are used as effectors in therapy or agriculture. For example, acircular polyribonucleotide made by the methods described herein (e.g.,the cell-free methods described herein) may be administered to a subject(e.g., in a pharmaceutical, veterinary, or agricultural composition). Inembodiments, the subject is a vertebrate animal (e.g., mammal, bird,fish, reptile, or amphibian). In embodiments, the subject is a human. Inembodiments, the method subject is a non-human mammal. In embodiments,the subject is a non-human mammal such as a non-human primate (e.g.,monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig,camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog,cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). Inembodiments, the subject is a bird, such as a member of the avian taxaGalliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes(e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus),Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).In embodiments, the subject is an invertebrate such as an arthropod(e.g, insects, arachnids, crustaceans), a nematode, an annelid, ahelminth, or a mollusc. In embodiments, the subject is an invertebrateagricultural pest or an invertebrate that is parasitic on aninvertebrate or vertebrate host. In embodiments, the subject is a plant,such as an angiosperm plant (which can be a dicot or a monocot) or agymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), afern, horsetail, clubmoss, or a bryophyte. In embodiments, the subjectis a eukaryotic alga (unicellular or multicellular). In embodiments, thesubject is a plant of agricultural or horticultural importance, such asrow crop plants, fruit-producing plants and trees, vegetables, trees,and ornamental plants including ornamental flowers, shrubs, trees,groundcovers, and turf grasses.

Secreted Polypeptide Effectors

In some embodiments, the circular polyribonucleotide described herein(e.g., the polyribonucleotide cargo of the circular polyribonucleotide)includes at least one coding sequence encoding a secreted polypeptideeffector. Exemplary secreted polypeptide effectors or proteins that maybe expressed include, e.g., cytokines and cytokine receptors,polypeptide hormones and receptors, growth factors, clotting factors,therapeutic replacement enzymes and therapeutic non-enzymatic effectors,regeneration, repair, and fibrosis factors, transformation factors, andproteins that stimulate cellular regeneration, non-limiting examples ofwhich are described herein, e.g., in the tables below.

Cytokines and Cytokine Receptors

In some embodiments, an effector described herein comprises a cytokineof Table 1, or a functional variant or fragment thereof, e.g., a proteinhaving at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a proteinsequence disclosed in Table 1 by reference to its UniProt ID. In someembodiments, the functional variant binds to the corresponding cytokinereceptor with a Kd of no more than 10%, 20%, 30%, 40%, or 50% higher orlower than the Kd of the corresponding wild-type cytokine for the samereceptor under the same conditions. In some embodiments, the effectorcomprises a fusion protein comprising a first region (e.g., a cytokinepolypeptide of Table 1 or a functional variant or fragment thereof) anda second, heterologous region. In some embodiments, the first region isa first cytokine polypeptide of Table 1. In some embodiments, the secondregion is a second cytokine polypeptide of Table 1, wherein the firstand second cytokine polypeptides form a cytokine heterodimer with eachother in a wild-type cell. In some embodiments, the polypeptide of Table1 or functional variant thereof comprises a signal sequence, e.g., asignal sequence that is endogenous to the effector, or a heterologoussignal sequence.

In some embodiments, an effector described herein comprises an antibodyor fragment thereof that binds a cytokine of Table 1. In someembodiments, the antibody molecule comprises a signal sequence.

TABLE 1 Exemplary cytokines and cytokine receptors Cytokine Cytokinereceptor(s) Entrez Gene ID¹ UniProt ID² IL-1α, IL-1β, or a heterodimerthereof IL-1 type 1 receptor, IL-1 type 2 receptor 3552, 3553 P01583,P01584 IL-1 Ra IL-1 type 1 receptor, IL-1 type 2 receptor 3454, 3455P17181, P48551 IL-2 IL-2R 3558 P60568 IL-3 IL-3 receptor α + β c (CD131)3562 P08700 IL-4 IL-4R type I, IL-4R type II 3565 P05112 IL-5 IL-5R 3567P05113 IL-6 IL-6R (sIL-6R) gp130 3569 P05231 IL-7 IL-7R and sIL-7R 3574P13232 IL-8 CXCR1 and CXCR2 3576 P10145 IL-9 IL-9R 3578 P15248 IL-10IL-10R1/IL-10R2 complex 3586 P22301 IL-11 IL-11 Rα 1 gp130 3589 P20809IL-12 (e.g., p35, p40, or a heterodimer thereof) IL-12Rβ1 and IL-12Rβ23593, 3592 P29459, P29460 IL-13 IL-13R1α1 and IL-13R1α2 3596 P35225IL-14 IL-14R 30685 P40222 IL-15 IL-15R 3600 P40933 IL-16 CD4 3603 Q14005IL-17A IL-17RA 3605 Q16552 IL-17B IL-17RB 27190 Q9UHF5 IL-17C IL-17RA toIL-17RE 27189 Q9P0M4 IL-17D SEF 53342 Q8TAD2 IL-17F IL-17RA, IL-17RC112744 Q96PD4 IL-18 IL-18 receptor 3606 Q14116 IL-19 IL-20R1/IL-20R229949 Q9UHD0 IL-20 L-20R1/IL-20R2 and IL-22R1/ IL-20R2 50604 Q9NYY1IL-21 IL-21R 59067 Q9HBE4 IL-22 IL-22R 50616 Q9GZX6 IL-23 (e.g., p19,p40, or a heterodimer thereof) IL-23R 51561 Q9NPF7 IL-24 IL-20R1/IL-20R2and IL-22R1/ IL-20R2 11009 Q13007 IL-25 IL-17RA and IL-17RB 64806 Q9H293IL-26 IL-10R2 chain and IL-20R1 chain 55801 Q9NPH9 IL-27 (e.g., p28,EBI3, or a heterodimer thereof) WSX-1 and gp130 246778 Q8NEV9 IL-28A,IL-28B, and IL29 IL-28R1/IL-10R2 282617, 282618 Q8IZI9, Q8IU54 IL-30IL6R/gp130 246778 Q8NEV9 IL-31 IL-31RA/OSMRβ 386653 Q6EBC2 IL-32 9235P24001 IL-33 ST2 90865 O95760 IL-34 Colony-stimulating factor 1 receptor146433 Q6ZMJ4 IL-35 (e.g., p35, EBI3, or a heterodimer thereof)IL-12Rβ2/gp130; IL-12Rβ2/IL-12Rβ2; gp130/gp130 10148 Q14213 IL-36IL-36Ra 27179 Q9UHA7 IL-37 IL-18Rα and IL-18BP 27178 Q9NZH6 IL-38IL-1R1, IL-36R 84639 Q8WWZ1 IFN-α IFNAR 3454 P17181 IFN-β IFNAR 3454P17181 IFN-γ IFNGR1/IFNGR2 3459 P15260 TGF-β TβR-I and TβR-II 7046, 7048P36897, P37173 TNF-α TNFR1, TNFR2 7132, 7133 P19438, P20333 ¹ Sequenceavailable on the NCBI database on the world wide web internet site“ncbi.nlm.nih.gov/gene”; Maglott D, et al. Gene: a gene-centeredinformation resource at NCBI. Nucleic Acids Res. 2014. pii: gku1055. ²Sequence available on the Uniprot database on the world wide webinternet site “uniprot.org/uniprot/”; UniProt: the universal proteinknowledgebase in 2021.Nucleic Acids Res. 49:D1 (2021).

Polypeptide Hormones and Receptors

In some embodiments, an effector described herein comprises a hormone ofTable 2, or a functional variant thereof, e.g., a protein having atleast 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequencedisclosed in Table 2 by reference to its UniProt ID. In someembodiments, the functional variant binds to the corresponding receptorwith a Kd of no more than 10%, 20%, 30%, 40%, or 50% higher than the Kdof the corresponding wild-type hormone for the same receptor under thesame conditions. In some embodiments, the polypeptide of Table 2 orfunctional variant thereof comprises a signal sequence, e.g., a signalsequence that is endogenous to the effector, or a heterologous signalsequence.

In some embodiments, an effector described herein comprises an antibodymolecule (e.g., an scFv) that binds a hormone of Table 2. In someembodiments, an effector described herein comprises an antibody molecule(e.g., an scFv) that binds a hormone receptor of Table 2. In someembodiments, the antibody molecule comprises a signal sequence.

TABLE 2 Exemplary polypeptide hormones and receptors Hormone ReceptorEntrez Gene ID¹ UniProt ID² Natriuretic Peptide, e.g., AtrialNatriuretic Peptide (ANP) NPRA, NPRB, NPRC 4878 P01160 Brain NatriureticPeptide (BNP) NPRA, NPRB 4879 P16860 C-type natriuretic peptide (CNP)NPRB 4880 P23582 Growth hormone (GH) GHR 2690 P10912 Prolactin (PRL)PRLR 5617 P01236 Thyroid-stimulating hormone (TSH) TSH receptor 7253P16473 Adrenocorticotropic hormone (ACTH) ACTH receptor 5443 P01189Follicle-stimulating hormone (FSH) FSHR 2492 P23945 Luteinizing hormone(LH) LHR 3973 P22888 Antidiuretic hormone (ADH) Vasopressin receptors,e.g., V2; AVPR1A; AVPR1B; AVPR3; AVPR2 554 P30518 Oxytocin OXTR 5020P01178 Calcitonin Calcitonin receptor (CT) 796 P01258 Parathyroidhormone (PTH) PTH1R and PTH2R 5741 P01270 Insulin Insulin receptor (IR)3630 P01308 Glucagon Glucagon receptor 2641 P01275 GIP GIPR 2695 P09681Fibroblast growth factor 19 (FGF19) FGFR4 9965 O95750 Fibroblast growthfactor 21 (FGF21) FGFR1c, 2c, 3c 26291 Q9NSA1 Fibroblast growth factor23 (FGF23) FGFR1, 2, 4 8074 Q9GZV9 Melanocyte-stimulating hormone(alpha- MSH) MC1R, MC4R, MC5R Melanocyte-stimulating hormone (beta- MSH)MC4R Melanocyte-stimulating hormone (gamma- MSH) MC1R, MC3R, MC4R, MC5RProopiomelanocortin POMC (alpha- beta-, gamma-, MSH precursor) MC1R,MC3R, MC4R, MC5R 5443 P01189 Glycoprotein hormones alpha chain (CGA)1081 P01215 Follicle-stimulating hormone beta (FSHB) FSHR 2488 P01225Leptin LEPR 3952 P41159 Ghrelin GHSR 51738 Q9UBU3 ¹ Sequence availableon the NCBI database on the world wide web internet site“ncbi.nlm.nih.gov/gene”, Maglott D, et al. Gene: a gene-centeredinformation resource at NCBI. Nucleic Acids Res. 2014. pii: gku1055. ²Sequence available on the Uniprot database on the world wide webinternet site “uniprot.org/uniprot/”; UniProt: the universal proteinknowledgebase in 2021.Nucleic Acids Res. 49:D1 (2021).

Growth Factors

In some embodiments, an effector described herein comprises a growthfactor of Table 3, or a functional variant thereof, e.g., a proteinhaving at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a proteinsequence disclosed in Table 3 by reference to its UniProt ID. In someembodiments, the functional variant binds to the corresponding receptorwith a Kd of no more than 10%, 20%, 30%, 40%, or 50% higher than the Kdof the corresponding wild-type growth factor for the same receptor underthe same conditions. In some embodiments, the polypeptide of Table 3 orfunctional variant thereof comprises a signal sequence, e.g., a signalsequence that is endogenous to the effector, or a heterologous signalsequence.

In some embodiments, an effector described herein comprises an antibodyor fragment thereof that binds a growth factor of Table 3. In someembodiments, an effector described herein comprises an antibody molecule(e.g., an scFv) that binds a growth factor receptor of Table 3. In someembodiments, the antibody molecule comprises a signal sequence.

TABLE 3 Exemplary growth factors PDGF family Entrez Gene ID¹ UniProt ID²PDGF (e.g., PDGF-1, PDGF-2, or a heterodimer thereof) PDGF receptor,e.g., PDGFRα, PDGFRβ 5156 P16234 CSF-1 CSF1R 1435 P09603 SCF CD117 3815P10721 VEGF family VEGF (e.g., isoforms VEGF 121, VEGF 165, VEGFR-1,VEGFR-2 2321 P17948 VEGF 189, and VEGF 206) VEGF-B VEGFR-1 2321 P17949VEGF-C VEGFR-2 and VEGFR-3 2324 P35916 PIGF VEGFR-1 5281 Q07326 EGFfamily EGF EGFR 1950 P01133 TGF-α EGFR 7039 P01135 amphiregulin EGFR 374P15514 HB-EGF EGFR 1839 Q99075 betacellulin EGFR, ErbB-4 685 P35070epiregulin EGFR, ErbB-4 2069 014944 Heregulin EGFR, ErbB-4 3084 Q02297FGF family FGF-1, FGF-2, FGF-3, FGFR1, FGFR2, P05230, P09038, FGF-4,FGF-5, FGF-6, FGFR3, and FGFR4 P11487, P08620, FGF-7, FGF-8, FGF-9 2246,2247, 2248, 2249, P12034, P10767, 2250, 2251, 2252, 2253, P21781,P55075, 2254 P31371 Insulin family Insulin IR 3630 P01308 IGF-I IGF-Ireceptor, IGF- II receptor 3479 P05019 IGF-II IGF-II receptor 3481P01344 HGF family HGF MET receptor 3082 P14210 MSP RON 4485 P26927Neurotrophin family NGF LNGFR, trkA 4803 P01138 BDNF trkB 627 P23560NT-3 trkA, trkB, trkC 4908 P20783 NT-4 trkA, trkB 4909 P34130 NT-5 trkA,trkB 4909 P34130 Angiopoietin family ANGPT1 HPK-6/TEK 284 Q15389 ANGPT2HPK-6/TEK 285 015123 ANGPT3 HPK-6/TEK 9068 095841 ANGPT4 HPK-6/TEK 51378Q9Y264 ANGPTL2 LILRB2 & integrin α5β1 23452 Q9UKU9 ANGPTL3 LPL 27329Q9Y5C1 ANGPTL4 51129 Q9BY76 ANGPTL8 PirB 55908 Q6UXH0 ¹ Sequenceavailable on the NCBI database on the world wide web internet site“ncbi.nlm.nih.gov/gene”, Maglott D, et al. Gene: a gene-centeredinformation resource at NCBI. Nucleic Acids Res. 2014. pii: gku1055. ²Sequence available on the Uniprot database on the world wide webinternet site “uniprot.org/uniprot/”; UniProt: the universal proteinknowledgebase in 2021.Nucleic Acids Res. 49:D1 (2021).

Clotting Factors

In some embodiments, an effector described herein comprises apolypeptide of Table 4, or a functional variant thereof, e.g., a proteinhaving at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a proteinsequence disclosed in Table 4 by reference to its UniProt ID. In someembodiments, the functional variant catalyzes the same reaction as thecorresponding wild-type protein, e.g., at a rate no less than 10%, 20%,30%, 40%, or 50% lower or higher than the wild-type protein. In someembodiments, the polypeptide of Table 4 or functional variant thereofcomprises a signal sequence, e.g., a signal sequence that is endogenousto the effector, or a heterologous signal sequence.

TABLE 4 Clotting-associated factors Effector Indication Entrez Gene ID¹UniProt ID² Factor I (fibrinogen) Afibrinogenomia 2243, 2266, 2244P02671, P02679, P02675 Factor II Factor II Deficiency 2147 P00734 FactorIX Hemophilia B 2158 P00740 Factor V Owren’s disease 2153 P12259 FactorVIII Hemophilia A 2157 P00451 Factor X Stuart-Prower Factor Deficiency2159 P00742 Factor XI Hemophilia C 2160 P03951 Factor XIII FibrinStabilizing factor deficiency 2162,2165 P00488, P05160 vWF vonWillebrand disease 7450 P04275 ¹ Sequence available on the NCBI databaseon the world wide web internet site “ncbi.nlm.nih.gov/gene”, Maglott D,et al. Gene: a gene-centered information resource at NCBI. Nucleic AcidsRes. 2014. pii: gku1055. ² Sequence available on the Uniprot database onthe world wide web internet site “uniprot.org/uniprot/”; UniProt: theuniversal protein knowledgebase in 2021.Nucleic Acids Res. 49:D1 (2021).

Therapeutic Replacement Enzymes

In some embodiments, an effector described herein comprises an enzyme ofTable 5, or a functional variant thereof, e.g., a protein having atleast 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequencedisclosed in Table 5 by reference to its UniProt ID. In someembodiments, the functional variant catalyzes the same reaction as thecorresponding wild-type protein, e.g., at a rate no less or no more than10%, 20%, 30%, 40%, or 50% lower than the wild-type protein.

TABLE 5 Exemplary enzymatic effectors for enzyme deficiency EffectorDeficiency Entrez Gene ID¹ UniProt ID² 3-methylcrotonyl-CoA carboxylase3-methylcrotonyl-CoA carboxylase deficiency 56922, 64087 Q96RQ3, Q9HCC0Acetyl-CoA-glucosaminide N-acetyltransferase Mucopolysaccharidosis MPSIII (Sanfilippo’s syndrome) Type III-C 138050 Q68CP4 ADAMTS13 ThromboticThrombocytopenic Purpura 11093 Q76LX8 adenine phosphoribosyltransferaseAdenine phosphoribosyltransferase deficiency 353 P07741 Adenosinedeaminase Adenosine deaminase deficiency 100 P00813 ADP-ribose proteinhydrolase Glutamyl ribose-5-phosphate storage disease 26119, 54936Q5SW96, Q9NX46 alpha glucosidase Glycogen storage disease type 2(Pompe’s disease) 2548 P10253 Arginase Familial hyperarginemia 383, 384P05089, P78540 Arylsulfatase A Metachromatic leukodystrophy 410 P15289Cathepsin K Pycnodysostosis 1513 P43235 Ceramidase Farber’s disease(lipogranulomatosis) 125981,340485, 55331 Q8TDN7, Q5QJU3, Q9NUN7Cystathionine B synthase Homocystinuria 875 P35520 Dolichol-P-mannosesynthase Congenital disorders of N-glycosylation CDG Ie 8813,54344O60762, Q9P2X0 Dolicho-P-Glc:Man9GlcNAc2-PP-dolichol glucosyltransferaseCongenital disorders of N-glycosylation CDG Ic 84920 Q5BKT4Dolicho-P-Man:Man5GlcNAc2-PP-dolichol mannosyltransferase Congenitaldisorders of N-glycosylation CDG Id 10195 Q92685Dolichyl-P-glucose:Glc-1-Man-9-GlcNAc-2-PP-dolichyl-α-3-glucosyltransferaseCongenital disorders of N-glycosylation CDG Ih 79053 Q9BVK2Dolichyl-P-mannose:Man-7-GlcNAc-2-PP-dolichyl-α-6-mannosyltransferaseCongenital disorders of N-glycosylation CDG Ig 79087 Q9BV10 Factor IIFactor II Deficiency 2147 P00734 Factor IX Hemophilia B 2158 P00740Factor V Owren’s disease 2153 P12259 Factor VIII Hemophilia A 2157P00451 Factor X Stuart-Prower Factor Deficiency 2159 P00742 Factor XIHemophilia C 2160 P03951 Factor XIII Fibrin Stabilizing factordeficiency 2162,2165 P00488, P05160 Galactosamine-6-sulfate sulfataseMucopolysaccharidosis MPS IV (Morquio’s syndrome) Type IV-A 2588 P34059Galactosylceramide β-galactosidase Krabbe’s disease 2581 P54803Ganglioside β-galactosidase GM1 gangliosidosis, generalized 2720 P16278Ganglioside β-galactosidase GM2 gangliosidosis 2720 P16278 Gangliosideβ-galactosidase Sphingolipidosis Type I 2720 P16278 Gangliosideβ-galactosidase Sphingolipidosis Type II (juvenile type) 2720 P16278Ganglioside β-galactosidase Sphingolipidosis Type III (adult type) 2720P16278 Glucosidase I Congenital disorders of N-glycosylation CDG IIb2548 P10253 Glucosylceramide β-glucosidase Gaucher’s disease 2629 P04062Heparan-S-sulfate sulfamidase Mucopolysaccharidosis MPS III(Sanfilippo’s syndrome) Type III-A 6448 P51688 homogentisate oxidaseAlkaptonuria 3081 Q93099 Hyaluronidase Mucopolysaccharidosis MPS IX(hyaluronidase deficiency) 3373, 8692, 8372, 23553 Q12794, Q12891,O43820, Q2M3T9 Iduronate sulfate sulfatase Mucopolysaccharidosis MPS II(Hunter’s syndrome) 3423 P22304 Lecithin-cholesterol acyltransferase(LCAT) Complete LCAT deficiency, Fish-eye disease, atherosclerosis,hypercholesterolemia 3931 606967 Lysine oxidase Glutaric acidemia type I4015 P28300 Lysosomal acid lipase Cholesteryl ester storage disease(CESD) 3988 P38571 Lysosomal acid lipase Lysosomal acid lipasedeficiency 3988 P38571 lysosomal acid lipase Wolman’s disease 3988P38571 Lysosomal pepstatin-insensitive peptidase Ceroid lipofuscinosisLate infantile form (CLN2, Jansky-Bielschowsky disease) 1200 014773Mannose (Man) phosphate (P) isomerase Congenital disorders ofN-glycosylation CDG Ib 4351 P34949Mannosyl-α-1,6-glycoprotein-β-1,2-N-acetylglucosminyltransferaseCongenital disorders of N-glycosylation CDG IIa 4247 Q10469Metalloproteinase-2 Winchester syndrome 4313 P08253 methylmalonyl-CoAmutase Methylmalonic acidemia (vitamin b12 non-responsive) 4594 P22033N-Acetyl galactosamine α-4-sulfate sulfatase (arylsulfatase B)Mucopolysaccharidosis MPS VI (Maroteaux-Lamy syndrome) 411 P15848N-acetyl-D-glucosaminidase Mucopolysaccharidosis MPS III (Sanfilippo’ssyndrome) Type III-B 4669 P54802 N-Acetyl-galactosaminidase Schindler’sdisease Type I (infantile severe form) 4668 P17050N-Acetyl-galactosaminidase Schindler’s disease Type II (Kanzaki disease,adult-onset form) 4668 P17050 N-Acetyl-galactosaminidase Schindler’sdisease Type III (intermediate form) 4668 P17050N-acetyl-glucosaminine-6-sulfate sulfatase Mucopolysaccharidosis MPS III(Sanfilippo’s syndrome) Type III-D 2799 P15586N-acetylglucosaminyl-1-phosphotransferase Mucolipidosis ML III(pseudo-Hurler’s polydystrophy) 79158 Q3T906N-Acetylglucosaminyl-1-phosphotransferase catalytic subunitMucolipidosis ML II (I-cell disease) 79158 Q3T906N-acetylglucosaminyl-1-phosphotransferase, substrate-recognition subunitMucolipidosis ML III (pseudo-Hurler’s polydystrophy) Type III-C 84572Q9UJJ9 N-Aspartylglucosaminidase Aspartylglucosaminuria 175 P20933Neuraminidase 1 (sialidase) Sialidosis 4758 Q99519 Palmitoyl-proteinthioesterase-1 Ceroid lipofuscinosis Adult form (CLN4, Kufs’ disease)5538 P50897 Palmitoyl-protein thioesterase-1 Ceroid lipofuscinosisInfantile form (CLN1, Santavuori-Haltia disease) 5538 P50897Phenylalanine hydroxylase Phenylketonuria 5053 P00439Phosphomannomutase-2 Congenital disorders of N-glycosylation CDG Ia(solely neurologic and neurologic-multivisceral forms) 5373 015305Porphobilinogen deaminase Acute Intermittent Porphyria 3145 P08397Purine nucleoside phosphorylase Purine nucleoside phosphorylasedeficiency 4860 P00491 pyrimidine 5′ nucleotidase Hemolytic anemiaand/or pyrimidine 5′ nucleotidase deficiency 51251 Q9H0P0Sphingomyelinase Niemann-Pick disease type A 6609 P17405Sphingomyelinase Niemann-Pick disease type B 6609 P17405 Sterol27-hydroxylase Cerebrotendinous xanthomatosis (cholestanol lipidosis)1593 Q02318 Thymidine phosphorylase Mitochondrial neurogastrointestinalencephalomyopathy (MNGIE) 1890 P19971 Trihexosylceramide α-galactosidaseFabry’s disease 2717 P06280 tyrosinase, e.g., OCA1 albinism, e.g.,ocular albinism 7299 P14679 UDP-GlcNAc:dolichyl-P NAcGlcphosphotransferase Congenital disorders of N-glycosylation CDG Ij 1798Q9H3H5 UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase,sialin Sialuria French type 10020 Q9Y223 Uricase Lesch-Nyhan syndrome,gout 391051 No protein uridine diphosphate glucuronyl-transferase (e.g.,UGT1A1) Crigler-Najjar syndrome 54658 P22309 α-1,2-MannosyltransferaseCongenital disorders of N-glycosylation CDG II (608776) 79796 Q9H6U8α-1,2-Mannosyltransferase Congenital disorders of N-glycosylation, typeI (pre-Golgi glycosylation defects) 79796 Q9H6U8α-1,3-Mannosyltransferase Congenital disorders of N-glycosylation CDG Ii440138 Q2TAA5 α-D-Mannosidase α-Mannosidosis, type I (severe) or II(mild) 10195 Q92685 α-L-Fucosidase Fucosidosis 4123 Q9NTJ4α-I-Iduronidase Mucopolysaccharidosis MPS I H/S (Hurler-Scheie syndrome)2517 P04066 α-I-Iduronidase Mucopolysaccharidosis MPS I-H (Hurler’ssyndrome) 3425 P35475 α-I-Iduronidase Mucopolysaccharidosis MPS I-S(Scheie’s syndrome) 3425 P35475 β-1,4-Galactosyltransferase Congenitaldisorders of N-glycosylation CDG IId 3425 P35475β-1,4-Mannosyltransferase Congenital disorders of N-glycosylation CDG Ik2683 P15291 β-D-Mannosidase β-Mannosidosis 56052 Q9BT22 β-GalactosidaseMucopolysaccharidosis MPS IV (Morquio’s syndrome) Type IV-B 4126 O00462β-Glucuronidase Mucopolysaccharidosis MPS VII (Sly’s syndrome) 2720P16278 β-Hexosaminidase A Tay-Sachs disease 2990 P08236 β-HexosaminidaseB Sandhoff’s disease 3073 P06865 ¹ Sequence available on the NCBIdatabase on the world wide web internet site “ncbi.nlm.nih.gov/gene”,Maglott D, et al. Gene: a gene-centered information resource at NCBI.Nucleic Acids Res. 2014. pii: gku1055. ² Sequence available on theUniprot database on the world wide web internet site“uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in2021.Nucleic Acids Res. 49:D1 (2021).

Other Non-Enzymatic Effectors

In some embodiments, a therapeutic polypeptide described hereincomprises a polypeptide of Table 6, or a functional variant thereof,e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99%identity to a protein sequence disclosed in Table 6 by reference to itsUniProt ID.

TABLE 6 Exemplary non-enzymatic effectors and corresponding indicationsEffector Indication Entrez Gene ID¹ UniProt ID² Survival motor neuronprotein (SMN) spinal muscular atrophy 6606 Q16637 Dystrophin musculardystrophy (e.g., Duchenne muscular dystrophy or Becker musculardystrophy) 1756 P11532 Complement protein, e.g., Complement factor C1Complement Factor I deficiency 3426 P05156 Complement factor H Atypicalhemolytic uremic syndrome 3075 P08603 Cystinosin (lysosomal cystinetransporter) Cystinosis 1497 060931 Epididymal secretory protein 1 (HE1;NPC2 protein) Niemann-Pick disease Type C2 10577 P61916 GDP-fucosetransporter-1 Congenital disorders of N-glycosylation CDG IIc(Rambam-Hasharon syndrome) 55343 Q96A29 GM2 activator protein GM2activator protein deficiency (Tay-Sachs disease AB variant, GM2A) 2760Q17900 Lysosomal transmembrane CLN3 protein Ceroid lipofuscinosisJuvenile form (CLN3, Batten disease, Vogt-Spielmeyer disease) 1207Q13286 Lysosomal transmembrane CLN5 protein Ceroid lipofuscinosisVariant late infantile form, Finnish type (CLN5) 1203 O75503 Naphosphate cotransporter, sialin Infantile sialic acid storage disorder26503 Q9NRA2 Na phosphate cotransporter, sialin Sialuria Finnish type(Salla disease) 26503 Q9NRA2 NPC1 protein Niemann-Pick disease TypeC1/Type D 4864 015118 Oligomeric Golgi complex-7 Congenital disorders ofN-glycosylation CDG IIe 91949 P83436 Prosaposin Prosaposin deficiency5660 P07602 Protective protein/cathepsin A (PPCA) Galactosialidosis(Goldberg’s syndrome, combined neuraminidase and β-galactosidasedeficiency) 5476 P10619 Protein involved in mannose-P-dolicholutilization Congenital disorders of N-glycosylation CDG If 9526 O75352Saposin B Saposin B deficiency (sulfatide activator deficiency) 5660P07602 Saposin C Saposin C deficiency (Gaucher’s activator deficiency)5660 P07602 Sulfatase-modifying factor-1 Mucosulfatidosis (multiplesulfatase deficiency) 285362 Q8NBK3 Transmembrane CLN6 protein Ceroidlipofuscinosis Variant late infantile form (CLN6) 54982 Q9NWW5Transmembrane CLN8 protein Ceroid lipofuscinosis Progressive epilepsywith intellectual disability 2055 Q9UBY8 vWF von Willebrand disease 7450P04275 Factor I (fibrinogen) Afibrinogenomia 2243, 2244, 2266 P02671,P02675, P02679 erythropoietin (hEPO) ¹ Sequence available on the NCBIdatabase on the world wide web internet site “ncbi.nlm.nih.gov/gene”,Maglott D, et al. Gene: a gene-centered information resource at NCBI.Nucleic Acids Res. 2014. pii: gku1055. ² Sequence available on theUniprot database on the world wide web internet site“uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in2021.Nucleic Acids Res. 49:D1 (2021).

Regeneration, Repair and Fibrosis Factors

Therapeutic polypeptides described herein also include growth factors,e.g., as disclosed in Table 7, or functional variants thereof, e.g., aprotein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to aprotein sequence disclosed in Table 7 by reference to its NCBI proteinaccession number. Also included are antibodies or fragments thereofagainst such growth factors, or miRNAs that promote regeneration andrepair.

TABLE 7 Exemplary Regeneration, Repair, and Fibrosis Factors Target NCBIGene accession #¹ NCBI Protein accession # ² VEGF-A NG_008732NP_001165094 NRG-1 NG_012005 NP_001153471 FGF2 NG_029067 NP_001348594FGF1 Gene ID:2246 NP_001341882 miR199-3p MIMAT0000232 n/a miR590-3pMIMAT0004801 n/a miR17-92 MI0000071 On the world wide web internet site“ncbi.nlm.nih.gov/pmc/articles/PMC2732113/figure/F1/” miR222 MI0000299n/a miR302-367 MIR302A And MIR367 On the world wide web internet site“ncbi.nlm.nih.gov/pmc/articles/PMC4400607/” ¹ Sequence available on theworld wide web internet site “ncbi.nlm.nih.gov/gene” (Maglott D, et al.Gene: a gene-centered information resource at NCBI. Nucleic Acids Res.2014. Pii: gku1055.) ² Sequence available on the world wide web internetsite “ncbi.nlm.nih.gov/protein/”

Transformation Factors

Therapeutic polypeptides described herein also include transformationfactors, e.g., protein factors that transform fibroblasts intodifferentiated cell e.g., factors disclosed in Table 8 or functionalvariants thereof, e.g., a protein having at least 80%, 85%, 90%, 95%,967%, 98%, 99% identity to a protein sequence disclosed in Table 8 byreference to its UniProt ID.

TABLE 8 Polypeptides indicated for organ repair by transformingfibroblasts Target NCBI Gene accession # ¹ NCBI Protein accession #²MESP1 Gene ID: 55897 EAX02066 ETS2 GeneID: 2114 NP_005230 HAND2 GeneID:9464 NP_068808 MYOCARDIN GeneID: 93649 NP_001139784 ESRRA Gene ID: 2101AAH92470 miR1 MI0000651 n/a miR133 MI000450 n/a TGFb GeneID: 7040NP_000651.3 WNT Gene ID: 7471 NP_005421 JAK Gene ID: 3716 NP_001308784NOTCH GeneID: 4851 XP_011517019 ¹ Sequence available on the world wideweb internet site “ncbi.nlm.nih.gov/gene” (Maglott D, et al. Gene: agene-centered information resource at NCBI. Nucleic Acids Res. 2014.Pii: gku1055.) ² Sequence available on the world wide web internet site“ncbi.nlm.nih.gov/protein/”

Proteins That Stimulate Cellular Regeneration

Therapeutic polypeptides described herein also include proteins thatstimulate cellular regeneration e.g., proteins disclosed in Table 9 orfunctional variants thereof, e.g., a protein having at least 80%, 85%,90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed inTable 9 by reference to its UniProt ID.

TABLE 9 Exemplary proteins that stimulate cellular regeneration TargetGene accession # ¹ Protein accession # ² MST1 NG_016454 NP_066278 STK30Gene ID:26448 NP_036103 MST2 Gene ID: 6788 NP_006272 SAV1 Gene ID: 60485NP_068590 LATS1 Gene ID: 9113 NP_004681 LATS2 Gene ID: 26524 NP_055387YAP1 NG_029530 NP_001123617 CDKN2b NG_023297 NP_004927 CDKN2a NG_007485NP_478102 ¹ Sequence available on the world wide web internet site“ncbi.nlm.nih.gov/gene” (Maglott D, et al. Gene: a gene-centeredinformation resource at NCBI. Nucleic Acids Res. 2014. Pii: gku1055.) ²Sequence available on the world wide web internet site“ncbi.nlm.nih.gov/protein/”

In some embodiments, the circular polyribonucleotide comprises one ormore expression sequences (coding sequences) and is configured forpersistent expression in a cell of a subject in vivo. In someembodiments, the circular polyribonucleotide is configured such thatexpression of the one or more expression sequences in the cell at alater time point is equal to or higher than an earlier time point. Insuch embodiments, the expression of the one or more expression sequencesmay be either maintained at a relatively stable level or may increaseover time. The expression of the expression sequences may be relativelystable for an extended period of time. For instance, in some cases, theexpression of the one or more expression sequences in the cell over atime period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or moredays does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,or 5%. In some cases, in some cases, the expression of the one or moreexpression sequences in the cell is maintained at a level that does notvary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% forat least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.

Plant-Modifying Polypeptides

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the polyribonucleotide) includes at leastone expression sequence encoding a plant-modifying polypeptide. Aplant-modifying polypeptide refers to a polypeptide that can alter thegenetic properties (e.g., increase gene expression, decrease geneexpression, or otherwise alter the nucleotide sequence of DNA or RNA),epigenetic properties, or physiological or biochemical properties of aplant in a manner that results in a change in the plant’s physiology orphenotype, e.g.,an increase or decrease in the plant’s fitness. In someembodiments, the polyribonucleotide encodes two, three, four, five, six,seven, eight, nine, ten or more different plant-modifying polypeptides,or multiple copies of one or more plant-modifying polypeptides. Aplant-modifying polypeptide may change the physiology or phenotype of,or increase or decrease the fitness of, a variety of plants, or can beone that effects such change(s) in one or more specific plants (e.g., aspecific species or genera of plants).

Examples of polypeptides that can be used herein can include an enzyme(e.g., a metabolic recombinase, a helicase, an integrase, a RNAse, aDNAse, or a ubiquitination protein), a pore-forming protein, a signalingligand, a cell penetrating peptide, a transcription factor, a receptor,an antibody, a nanobody, a gene editing protein (e.g., CRISPR-Casendonuclease, TALEN, or zinc finger), riboprotein, a protein aptamer, ora chaperone.

Agricultural Polypeptides

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the polyribonucleotide) includes at leastone expression sequence encoding an agricultural polypeptide. Anagricultural polypeptide is a polypeptide that is suitable for anagricultural use. In embodiments, an agricultural polypeptide is appliedto a plant or seed (e.g., by foliar spray, dusting, injection, or seedcoating) or to the plant’s environment (e.g., by soil drench or granularsoil application), resulting in an alteration of the plant’s physiology,phenotype, or fitness. Embodiments of an agricultural polypeptideinclude polypeptides that alter a level, activity, or metabolism of oneor more microorganisms resident in or on a plant or non-human animalhost, the alteration resulting in an increase in the host’s fitness. Insome embodiments the agricultural polypeptide is a plant polypeptide. Insome embodiments, the agricultural polypeptide is an insect polypeptide.In some embodiments, the agricultural polypeptide has a biologicaleffect when contacted with a non-human vertebrate animal, invertebrateanimal, microbial, or plant cell.

In some embodiments, the polyribonucleotide encodes two, three, four,five, six, seven, eight, nine, ten or more agricultural polypeptides, ormultiple copies of one or more agricultural polypeptides.

Embodiments of polypeptides useful in agricultural applications include,for example, bacteriocins, lysins, antimicrobial peptides, nodule C-richpeptides, and bacteriocyte regulatory peptides. Such polypeptides can beused to alter the level, activity, or metabolism of targetmicroorganisms for increasing the fitness of insects, such as honeybeesand silkworms. Embodiments of agriculturally useful polypeptides includepeptide toxins, such as those naturally produced by entomopathogenicbacteria (e.g., Bacillus thuringiensis, Photorhabdus luminescens,Serratia entomophila, or Xenorhabdus nematophila), as is known in theart. Embodiments of agriculturally useful polypeptides includepolypeptides (including small peptides such as cyclodipeptides ordiketopiperazines) for controlling agriculturally important pests orpathogens, e.g., antimicrobial polypeptides or antifungal polypeptidesfor controlling diseases in plants, or pesticidal polypeptides (e.g.,insecticidal polypeptides or nematicidal polypeptides) for controllinginvertebrate pests such as insects or nematodes. Embodiments ofagriculturally useful polypeptides include antibodies, nanobodies, andfragments thereof, e.g., antibody or nanobody fragments that retain atleast some (e.g., at least 10%) of the specific binding activity of theintact antibody or nanobody. Embodiments of agriculturally usefulpolypeptides include transcription factors, e.g., plant transcriptionfactors; see., e.g, the “AtTFDB” database listing the transcriptionfactor families identified in the model plant Arabidopsis thaliana),publicly available at agris-knowledgebase[dot]org/AtTFDB/. Embodimentsof agriculturally useful polypeptides include nucleases, for example,exonucleases or endonucleases (e.g., Cas nucleases such as Cas9 orCas12a). Embodiments of agriculturally useful polypeptides furtherinclude cell-penetrating peptides, enzymes (e.g., amylases, cellulases,peptidases, lipases, chitinases), peptide pheromones (for example, yeastmating pheromones, invertebrate reproductive and larval signallingpheromones, see, e.g., Altstein (2004) Peptides, 25:1373-1376).

Internal Ribosomal Entry Sites

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the polyribonucleotide) includes one or moreinternal ribosome entry site (IRES) elements. In some embodiments, theIRES is operably linked to one or more expression sequences (e.g., eachIRES is operably linked to one or more expression sequences). Inembodiments, the IRES is located between a heterologous promoter and the5′ end of a coding sequence.

A suitable IRES element to include in a polyribonucleotide includes anRNA sequence capable of engaging a eukaryotic ribosome. In someembodiments, the IRES element is at least about 5 nt, at least about 8nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, atleast about 20 nt, at least about 25 nt, at least about 30 nt, at leastabout 40 nt, at least about 50 nt, at least about 100 nt, at least about200 nt, at least about 250 nt, at least about 350 nt, or at least about500 nt.

In some embodiments, the IRES element is derived from the DNA of anorganism including, but not limited to, a virus, a mammal, and aDrosophila. Such viral DNA may be derived from, but is not limited to,picornavirus complementary DNA (cDNA), with encephalomyocarditis virus(EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA fromwhich an IRES element is derived includes, but is not limited to, anAntennapedia gene from Drosophila melanogaster.

In some embodiments, if present, the IRES sequence is an IRES sequenceof Taura syndrome virus, Triatoma virus, Theiler’s encephalomyelitisvirus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padivirus, Reticuloendotheliosis virus, fuman poliovirus 1, Plautia stallintestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodiscacoagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodiscacoagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis Avirus, Hepatitis GB virus, foot and mouth disease virus, Humanenterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-likevirus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifertobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1,Black Queen Cell Virus, Aphid lethal paralysis virus, Avianencephalomyelitis virus, Acute bee paralysis virus, Hibiscus chloroticringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1,Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, HumanBAG-I, Human BCL2, Human BiP, Human c-IAPI , Human c-myc, Human eIF4G,Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx,Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-I, Mouse Rbm3,Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, MouseUtrA, Human VEGF-A, Human XIAP, Salivirus, Cosavirus, Parechovirus,Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Humanc-src, Human FGF-I, Simian picomavirus, Turnip crinkle virus, an aptamerto eIF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB½). In yetanother embodiment, the IRES is an IRES sequence of Coxsackievirus B3(CVB3). In a further embodiment, the IRES is an IRES sequence ofEncephalomyocarditis virus.

In some embodiments, the polyribonucleotide includes at least one IRESflanking at least one (e.g., 2, 3, 4, 5 or more) expression sequence. Insome embodiments, the IRES flanks both sides of at least one (e.g., 2,3, 4, 5 or more) expression sequence. In some embodiments, thepolyribonucleotide includes one or more IRES sequences on one or bothsides of each expression sequence, leading to separation of theresulting peptide(s) and or polypeptide(s).

In some embodiments, the polyribonucleotide cargo includes an IRES. Forexample, the polyribonucleotide cargo may include a circular RNA IRES,e.g., as described in Chen et al. Mol. Cell 81:1-19, 2021, which ishereby incorporated by reference in its entirety.

Regulatory Elements

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the polyribonucleotide) includes one or moreregulatory elements. In some embodiments, the polyribonucleotideincludes a regulatory element, e.g., a sequence that modifies expressionof an expression sequence within the polyribonucleotide.

A regulatory element may include a sequence that is located adjacent toan expression sequence that encodes an expression product. A regulatoryelement may be linked operatively to the adjacent sequence. A regulatoryelement may increase an amount of product expressed as compared to anamount of the expressed product when no regulatory element exists. Inaddition, one regulatory element can increase an amount of productsexpressed for multiple expression sequences attached in tandem. Hence,one regulatory element can enhance the expression of one or moreexpression sequences. Multiple regulatory elements are well-known topersons of ordinary skill in the art.

In some embodiments, the regulatory element is a translation modulator.A translation modulator can modulate translation of the expressionsequence in the polyribonucleotide. A translation modulator can be atranslation enhancer or suppressor. In some embodiments, thepolyribonucleotide includes at least one translation modulator adjacentto at least one expression sequence. In some embodiments, thepolyribonucleotide includes a translation modulator adjacent eachexpression sequence. In some embodiments, the translation modulator ispresent on one or both sides of each expression sequence, leading toseparation of the expression products, e.g., peptide(s) and orpolypeptide (s).

In some embodiments, the regulatory element is a microRNA (miRNA) or amiRNA binding site.

Further examples of regulatory elements are described, e.g., inparagraphs [0154] - [0161] of International Patent Publication No.WO2019/118919, which is hereby incorporated by reference in itsentirety.

Translation Initiation Sequences

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the polyribonucleotide) includes at leastone translation initiation sequence. In some embodiments, thepolyribonucleotide includes a translation initiation sequence operablylinked to an expression sequence.

In some embodiments, the polyribonucleotide encodes a polypeptide andmay include a translation initiation sequence, e.g., a start codon. Insome embodiments, the translation initiation sequence includes a Kozakor Shine-Dalgamo sequence. In some embodiments, the polyribonucleotideincludes the translation initiation sequence, e.g., Kozak sequence,adjacent to an expression sequence. In some embodiments, the translationinitiation sequence is a non-coding start codon. In some embodiments,the translation initiation sequence, e.g., Kozak sequence, is present onone or both sides of each expression sequence, leading to separation ofthe expression products. In some embodiments, the polyribonucleotideincludes at least one translation initiation sequence adjacent to anexpression sequence. In some embodiments, the translation initiationsequence provides conformational flexibility to the polyribonucleotide.In some embodiments, the translation initiation sequence is within asubstantially single stranded region of the polyribonucleotide. Furtherexamples of translation initiation sequences are described in paragraphs[0163] - [0165] of International Patent Publication No. WO2019/118919,which is hereby incorporated by reference in its entirety.

The polyribonucleotide may include more than 1 start codon such as, butnot limited to, at least 2, at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 50, at least 60 or more than 60 startcodons. Translation may initiate on the first start codon or mayinitiate downstream of the first start codon.

In some embodiments, the polyribonucleotide may initiate at a codonwhich is not the first start codon, e.g., AUG. Translation of thepolyribonucleotide may initiate at an alternative translation initiationsequence, such as, but not limited to, ACG, AGG, AAG, CTG/CUG (SEQ IDNO: 74), GTG/GUG (SEQ ID NO: 75), ATA/AUA (SEQ ID NO: 76), ATT/AUU (SEQID NO: 77), TTG/UUG (SEQ ID NO: 78). In some embodiments, translationbegins at an alternative translation initiation sequence under selectiveconditions, e.g., stress induced conditions. As a non-limiting example,the translation of the polyribonucleotide may begin at alternativetranslation initiation sequence, such as ACG. As another non-limitingexample, the polyribonucleotide translation may begin at alternativetranslation initiation sequence, CTG/CUG (SEQ ID NO: 74). As anothernon-limiting example, the polyribonucleotide translation may begin atalternative translation initiation sequence, GTG/GUG (SEQ ID NO: 75). Asanother non-limiting example, the polyribonucleotide may begintranslation at a repeat-associated non-AUG (RAN) sequence, such as analternative translation initiation sequence that includes shortstretches of repetitive RNA e.g., CGG, GGGGCC (SEQ DI NO: 79), CAG, CTG.

Termination Elements

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the polyribonucleotide) includes least onetermination element. In some embodiments, the polyribonucleotideincludes a termination element operably linked to an expressionsequence. In some embodiments, the polynucleotide lacks a terminationelement.

In some embodiments, the polyribonucleotide includes one or moreexpression sequences, and each expression sequence may or may not have atermination element. In some embodiments, the polyribonucleotideincludes one or more expression sequences, and the expression sequenceslack a termination element, such that the polyribonucleotide iscontinuously translated. Exclusion of a termination element may resultin rolling circle translation or continuous expression of expressionproduct.

In some embodiments, the circular polyribonucleotide includes one ormore expression sequences, and each expression sequence may or may nothave a termination element. In some embodiments, the circularpolyribonucleotide includes one or more expression sequences, and theexpression sequences lack a termination element, such that the circularpolyribonucleotide is continuously translated. Exclusion of atermination element may result in rolling circle translation orcontinuous expression of expression product, e.g., peptides orpolypeptides, due to lack of ribosome stalling or fall-off. In such anembodiment, rolling circle translation expresses a continuous expressionproduct through each expression sequence. In some other embodiments, atermination element of an expression sequence can be part of a staggerelement. In some embodiments, one or more expression sequences in thecircular polyribonucleotide comprises a termination element. However,rolling circle translation or expression of a succeeding (e.g., second,third, fourth, fifth, etc.) expression sequence in the circularpolyribonucleotide is performed. In such instances, the expressionproduct may fall off the ribosome when the ribosome encounters thetermination element, e.g., a stop codon, and terminates translation. Insome embodiments, translation is terminated while the ribosome, e.g., atleast one subunit of the ribosome, remains in contact with the circularpolyribonucleotide.

In some embodiments, the circular polyribonucleotide includes atermination element at the end of one or more expression sequences. Insome embodiments, one or more expression sequences comprises two or moretermination elements in succession. In such embodiments, translation isterminated and rolling circle translation is terminated. In someembodiments, the ribosome completely disengages with the circularpolyribonucleotide. In some such embodiments, production of a succeeding(e.g., second, third, fourth, fifth, etc.) expression sequence in thecircular polyribonucleotide may require the ribosome to reengage withthe circular polyribonucleotide prior to initiation of translation.Generally, termination elements include an in-frame nucleotide tripletthat signals termination of translation, e.g., UAA, UGA, UAG. In someembodiments, one or more termination elements in the circularpolyribonucleotide are frame-shifted termination elements, such as butnot limited to, off-frame or -1 and + 1 shifted reading frames (e.g.,hidden stop) that may terminate translation. Frame-shifted terminationelements include nucleotide triples, TAA, TAG, and TGA that appear inthe second and third reading frames of an expression sequence.Frame-shifted termination elements may be important in preventingmisreads of mRNA, which is often detrimental to the cell. In someembodiments, the termination element is a stop codon.

Further examples of termination elements are described in paragraphs[0169] - [0170] of International Patent Publication No. WO2019/118919,which is hereby incorporated by reference in its entirety.

Untranslated Regions

In some embodiments, a circular polyribonucleotide includes untranslatedregions (UTRs). UTRs of a genomic region including a gene may betranscribed but not translated. In some embodiments, a UTR may beincluded upstream of the translation initiation sequence of anexpression sequence described herein. In some embodiments, a UTR may beincluded downstream of an expression sequence described herein. In someinstances, one UTR for first expression sequence is the same as orcontinuous with or overlapping with another UTR for a second expressionsequence. In some embodiments, the intron is a human intron. In someembodiments, the intron is a full-length human intron, e.g., ZKSCAN1.

Exemplary untranslated regions are described in paragraphs [0197] -[201] of International Patent Publication No. WO2019/118919, which ishereby incorporated by reference in its entirety.

In some embodiments, a circular polyribonucleotide includes a poly-Asequence. Exemplary poly-A sequences are described in paragraphs[0202] - [0205] of International Patent Publication No. WO2019/118919,which is hereby incorporated by reference in its entirety. In someembodiments, a circular polyribonucleotide lacks a poly-A sequence.

In some embodiments, a circular polyribonucleotide includes a UTR withone or more stretches of Adenosines and Uridines embedded within. TheseAU rich signatures may increase turnover rates of the expressionproduct.

Introduction, removal, or modification of UTR AU rich elements (AREs)may be useful to modulate the stability, or immunogenicity (e.g., thelevel of one or more marker of an immune or inflammatory response) ofthe circular polyribonucleotide. When engineering specific circularpolyribonucleotides, one or more copies of an ARE may be introduced tothe circular polyribonucleotide and the copies of an ARE may modulatetranslation and/or production of an expression product. Likewise, AREsmay be identified and removed or engineered into the circularpolyribonucleotide to modulate the intracellular stability and thusaffect translation and production of the resultant protein.

It should be understood that any UTR from any gene may be incorporatedinto the respective flanking regions of the circular polyribonucleotide.

In some embodiments, a circular polyribonucleotide lacks a 5′-UTR and iscompetent for protein expression from its one or more expressionsequences. In some embodiments, the circular polyribonucleotide lacks a3′-UTR and is competent for protein expression from its one or moreexpression sequences. In some embodiments, the circularpolyribonucleotide lacks a poly-A sequence and is competent for proteinexpression from its one or more expression sequences. In someembodiments, the circular polyribonucleotide lacks a termination elementand is competent for protein expression from its one or more expressionsequences. In some embodiments, the circular polyribonucleotide lacks aninternal ribosomal entry site and is competent for protein expressionfrom its one or more expression sequences. In some embodiments, thecircular polyribonucleotide lacks a cap and is competent for proteinexpression from its one or more expression sequences. In someembodiments, the circular polyribonucleotide lacks a 5′-UTR, a 3′-UTR,and an IRES, and is competent for protein expression from its one ormore expression sequences. In some embodiments, the circularpolyribonucleotide includes one or more of the following sequences: asequence that encodes one or more miRNAs, a sequence that encodes one ormore replication proteins, a sequence that encodes an exogenous gene, asequence that encodes a therapeutic, a regulatory element (e.g.,translation modulator, e.g., translation enhancer or suppressor), atranslation initiation sequence, one or more regulatory nucleic acidsthat targets endogenous genes (e.g., siRNA, IncRNAs, shRNA), and asequence that encodes a therapeutic mRNA or protein.

In some embodiments, a circular polyribonucleotide lacks a 5′-UTR. Insome embodiments, the circular polyribonucleotide lacks a 3′-UTR. Insome embodiments, the circular polyribonucleotide lacks a poly-Asequence. In some embodiments, the circular polyribonucleotide lacks atermination element. In some embodiments, the circularpolyribonucleotide lacks an internal ribosomal entry site. In someembodiments, the circular polyribonucleotide lacks degradationsusceptibility by exonucleases. In some embodiments, the fact that thecircular polyribonucleotide lacks degradation susceptibility can meanthat the circular polyribonucleotide is not degraded by an exonuclease,or only degraded in the presence of an exonuclease to a limited extent,e.g., that is comparable to or similar to in the absence of exonuclease.In some embodiments, the circular polyribonucleotide is not degraded byexonucleases. In some embodiments, the circular polyribonucleotide hasreduced degradation when exposed to exonuclease. In some embodiments,the circular polyribonucleotide lacks binding to a cap-binding protein.In some embodiments, the circular polyribonucleotide lacks a 5′ cap.

Stagger Elements

In some embodiments, the circular polyribonucleotide includes at leastone stagger element adjacent to an expression sequence. In someembodiments, the circular polyribonucleotide includes a stagger elementadjacent to each expression sequence. In some embodiments, the staggerelement is present on one or both sides of each expression sequence,leading to separation of the expression products, e.g., peptide(s) andor polypeptide(s). In some embodiments, the stagger element is a portionof the one or more expression sequences. In some embodiments, thecircular polyribonucleotide comprises one or more expression sequences,and each of the one or more expression sequences is separated from asucceeding expression sequence by a stagger element on the circularpolyribonucleotide. In some embodiments, the stagger element preventsgeneration of a single polypeptide (a) from two rounds of translation ofa single expression sequence or (b) from one or more rounds oftranslation of two or more expression sequences. In some embodiments,the stagger element is a sequence separate from the one or moreexpression sequences. In some embodiments, the stagger element comprisesa portion of an expression sequence of the one or more expressionsequences.

In some embodiments, the circular polyribonucleotide includes a staggerelement. To avoid production of a continuous expression product, e.g.,peptide or polypeptide, while maintaining rolling circle translation, astagger element may be included to induce ribosomal pausing duringtranslation. In some embodiments, the stagger element is at 3′ end of atleast one of the one or more expression sequences. The stagger elementcan be configured to stall a ribosome during rolling circle translationof the circular polyribonucleotide. The stagger element may include, butis not limited to a 2A-like, or CHYSEL (SEQ ID NO: 71) (cis-actinghydrolase element) sequence. In some embodiments, the stagger elementencodes a sequence with a C-terminal consensus sequence that isX₁X₂X₃EX₅NPGP (SEQ ID NO: 72), where X₁ is absent or G or H, X₂ isabsent or D or G, X₃ is D or V or I or S or M, and X₅ is any amino acid.In some embodiments, this sequence comprises a non-conserved sequence ofamino-acids with a strong alpha-helical propensity followed by theconsensus sequence -D(V/I)EXNPGP (SEQ ID NO: 73), where x= any aminoacid. Some nonlimiting examples of stagger elements includes GDVESNPGP(SEQ ID NO: 52), GDIEENPGP (SEQ ID NO: 53), VEPNPGP (SEQ ID NO: 54),IETNPGP (SEQ ID NO: 55), GDIESNPGP (SEQ ID NO: 56), GDVELNPGP (SEQ IDNO: 57), GDIETNPGP (SEQ ID NO: 58), GDVENPGP (SEQ ID NO: 59), GDVEENPGP(SEQ ID NO: 60), GDVEQNPGP (SEQ ID NO: 61), IESNPGP (SEQ ID NO: 62),GDIELNPGP (SEQ ID NO: 63), HDIETNPGP (SEQ ID NO: 64), HDVETNPGP (SEQ IDNO: 65), HDVEMNPGP (SEQ ID NO: 66), GDMESNPGP (SEQ ID NO: 67), GDVETNPGP(SEQ ID NO: 68) GDIEQNPGP (SEQ ID NO: 69), and DSEFNPGP (SEQ ID NO: 70).

In some embodiments, the stagger element described herein cleaves anexpression product, such as between G and P of the consensus sequencedescribed herein. As one non-limiting example, the circularpolyribonucleotide includes at least one stagger element to cleave theexpression product. In some embodiments, the circular polyribonucleotideincludes a stagger element adjacent to at least one expression sequence.In some embodiments, the circular polyribonucleotide includes a staggerelement after each expression sequence. In some embodiments, thecircular polyribonucleotide includes a stagger element is present on oneor both sides of each expression sequence, leading to translation ofindividual peptide(s) and or polypeptide(s) from each expressionsequence.

In some embodiments, a stagger element comprises one or more modifiednucleotides or unnatural nucleotides that induce ribosomal pausingduring translation. Unnatural nucleotides may include peptide nucleicacid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycolnucleic acid (GNA) and threose nucleic acid (TNA). Examples such asthese are distinguished from naturally occurring DNA or RNA by changesto the backbone of the molecule. Exemplary modifications can include anymodification to the sugar, the nucleobase, the intemucleoside linkage(e.g., to a linking phosphate / to a phosphodiester linkage / to thephosphodiester backbone), and any combination thereof that can induceribosomal pausing during translation. Some of the exemplarymodifications provided herein are described elsewhere herein.

In some embodiments, the stagger element is present in the circularpolyribonucleotide in other forms. For example, in some exemplarycircular polyribonucleotides, a stagger element comprises a terminationelement of a first expression sequence in the circularpolyribonucleotide, and a nucleotide spacer sequence that separates thetermination element from a first translation initiation sequence of anexpression succeeding the first expression sequence. In some examples,the first stagger element of the first expression sequence is upstreamof (5′ to) a first translation initiation sequence of the expressionsucceeding the first expression sequence in the circularpolyribonucleotide. In some cases, the first expression sequence and theexpression sequence succeeding the first expression sequence are twoseparate expression sequences in the circular polyribonucleotide. Thedistance between the first stagger element and the first translationinitiation sequence can enable continuous translation of the firstexpression sequence and its succeeding expression sequence.

In some embodiments, the first stagger element comprises a terminationelement and separates an expression product of the first expressionsequence from an expression product of its succeeding expressionsequences, thereby creating discrete expression products. In some cases,the circular polyribonucleotide comprising the first stagger elementupstream of the first translation initiation sequence of the succeedingsequence in the circular polyribonucleotide is continuously translated,while a corresponding circular polyribonucleotide comprising a staggerelement of a second expression sequence that is upstream of a secondtranslation initiation sequence of an expression sequence succeeding thesecond expression sequence is not continuously translated. In somecases, there is only one expression sequence in the circularpolyribonucleotide, and the first expression sequence and its succeedingexpression sequence are the same expression sequence. In some exemplarycircular polyribonucleotides, a stagger element comprises a firsttermination element of a first expression sequence in the circularpolyribonucleotide, and a nucleotide spacer sequence that separates thetermination element from a downstream translation initiation sequence.In some such examples, the first stagger element is upstream of (5′ to)a first translation initiation sequence of the first expression sequencein the circular polyribonucleotide. In some cases, the distance betweenthe first stagger element and the first translation initiation sequenceenables continuous translation of the first expression sequence and anysucceeding expression sequences.

In some embodiments, the first stagger element separates one roundexpression product of the first expression sequence from the next roundexpression product of the first expression sequences, thereby creatingdiscrete expression products. In some cases, the circularpolyribonucleotide comprising the first stagger element upstream of thefirst translation initiation sequence of the first expression sequencein the circular polyribonucleotide is continuously translated, while acorresponding circular polyribonucleotide comprising a stagger elementupstream of a second translation initiation sequence of a secondexpression sequence in the corresponding circular polyribonucleotide isnot continuously translated. In some cases, the distance between thesecond stagger element and the second translation initiation sequence isat least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x greater in thecorresponding circular polyribonucleotide than a distance between thefirst stagger element and the first translation initiation in thecircular polyribonucleotide. In some cases, the distance between thefirst stagger element and the first translation initiation is at least 2nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater.In some embodiments, the distance between the second stagger element andthe second translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt,17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance between thefirst stagger element and the first translation initiation. In someembodiments, the circular polyribonucleotide comprises more than oneexpression sequence.

Examples of stagger elements are described in paragraphs [0172] - [0175]of International Patent Publication No. WO2019/118919, which is herebyincorporated by reference in its entirety.

Non-Coding Sequences

In some embodiments, the polyribonucleotide described herein (e.g., thepolyribonucleotide cargo of the polyribonucleotide) includes one or morenon-coding sequence, e.g., a sequence that does not encode theexpression of polypeptide. In some embodiments, the polyribonucleotideincludes two, three, four, five, six, seven, eight, nine, ten or morethan ten non-coding sequences. In some embodiments, thepolyribonucleotide does not encode a polypeptide expression sequence.

Noncoding sequences can be natural or synthetic sequences. In someembodiments, a noncoding sequence can alter cellular behavior, such ase.g., lymphocyte behavior. In some embodiments, the noncoding sequencesare antisense to cellular RNA sequences.

In some embodiments, the polyribonucleotide includes regulatory nucleicacids that are RNA or RNA-like structures typically from about 5-500base pairs (bp) (depending on the specific RNA structure (e.g., miRNA5-30 bp, IncRNA 200-500 bp) and may have a nucleobase sequence identical(complementary) or nearly identical (substantially complementary) to acoding sequence in an expressed target gene within the cell. Inembodiments, the circular polyribonucleotide includes regulatory nucleicacids that encode an RNA precursor that can be processed to a smallerRNA, e.g., a miRNA precursor, which can be from about 50 to about 1000bp, that can be processed to a smaller miRNA intermediate or a maturemiRNA.

Long non-coding RNAs (IncRNA) are defined as non-protein codingtranscripts longer than 100 nucleotides. Many IncRNAs are characterizedas tissue specific. Divergent IncRNAs that are transcribed in theopposite direction to nearby protein-coding genes include a significantproportion (e.g., about 20% of total IncRNAs in mammalian genomes) andpossibly regulate the transcription of the nearby gene. In oneembodiment, the polyribonucleotide provided herein includes a sensestrand of a IncRNA. In one embodiment, the polyribonucleotide providedherein includes an antisense strand of a IncRNA.

In embodiments, the polyribonucleotide encodes a regulatory nucleic acidthat is substantially complementary, or fully complementary, to all orto at least one fragment of an endogenous gene or gene product (e.g.,mRNA). In embodiments, the regulatory nucleic acids complement sequencesat the boundary between introns and exons, in between exons, or adjacentto an exon, to prevent the maturation of newly generated nuclear RNAtranscripts of specific genes into mRNA for transcription. Theregulatory nucleic acids that are complementary to specific genes canhybridize with the mRNA for that gene and prevent its translation. Theantisense regulatory nucleic acid can be DNA, RNA, or a derivative orhybrid thereof. In some embodiments, the regulatory nucleic acidincludes a protein-binding site that can bind to a protein thatparticipates in regulation of expression of an endogenous gene or anexogenous gene.

In embodiments, the polyribonucleotide encodes a regulatory RNA thathybridizes to a transcript of interest wherein the regulatory RNA has alength of from about 5 to 30 nucleotides, from about 10 to 30nucleotides, or about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides. Inembodiments, the degree of sequence identity of the regulatory RNA tothe targeted transcript is at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%.

In embodiments, the polyribonucleotide encodes a microRNA (miRNA)molecule identical to about 5 to about 25 contiguous nucleotides of atarget gene or encodes a precursor to that miRNA. In some embodiments,the miRNA has a sequence that allows the mRNA to recognize and bind to aspecific target mRNA. In embodiments, miRNA sequence commences with thedinucleotide AA, includes a GC -content of about 30-70% (about 30-60%,about 40-60%, or about 45%-55%), and does not have a high percentageidentity to any nucleotide sequence other than the target in the genomeof the subject (e.g., a mammal) in which it is to be introduced, forexample as determined by standard BLAST search.

In some embodiments, the polyribonucleotide includes at least one miRNA(or miRNA precursor), e.g., 2, 3, 4, 5, 6, or more miRNAs or miRNAprecursors. In some embodiments, the polyribonucleotide includes asequence that encodes a miRNA (or its precursor) having at least about75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, or 99% or 100% nucleotidesequence complementarity to a target sequence.

siRNAs and shRNAs resemble intermediates in the processing pathway ofthe endogenous microRNA (miRNA) genes. In some embodiments, siRNAs canfunction as miRNAs and vice versa. MicroRNAs, like siRNAs, use RISC todownregulate target genes, but unlike siRNAs, most animal miRNAs do notcleave the mRNA. Instead, miRNAs reduce protein output throughtranslational suppression or polyA removal and mRNA degradation. KnownmiRNA binding sites are within mRNA 3′ UTRs; miRNAs seem to target siteswith near-perfect complementarity to nucleotides 2-8 from the miRNA’s 5′end. This region is known as the seed region. Because mature siRNAs andmiRNAs are interchangeable, exogenous siRNAs downregulate mRNAs withseed complementarity to the siRNA.

Lists of known miRNA sequences can be found in databases maintained byresearch organizations, such as Wellcome Trust Sanger Institute, PennCenter for Bioinformatics, Memorial Sloan Kettering Cancer Center, andEuropean Molecule Biology Laboratory, among others. Known effectivesiRNA sequences and cognate binding sites are also well represented inthe relevant literature. RNAi molecules are readily designed andproduced by technologies known in the art. In addition, there arecomputational tools that increase the chance of finding effective andspecific sequence motifs.

Protein-Binding Sequences

In some embodiments, a circular polyribonucleotide includes one or moreprotein binding sites that enable a protein, e.g., a ribosome, to bindto an internal site in the RNA sequence. By engineering protein bindingsites, e.g., ribosome binding sites, into the circularpolyribonucleotide, the circular polyribonucleotide may evade or havereduced detection by the host’s immune system, have modulateddegradation, or modulated translation, by masking the circularpolyribonucleotide from components of the host’s immune system.

In some embodiments, a circular polyribonucleotide includes at least oneimmunoprotein binding site, for example to evade immune responses, e.g.,CTL (cytotoxic T lymphocyte) responses. In some embodiments, theimmunoprotein binding site is a nucleotide sequence that binds to animmunoprotein and aids in masking the circular polyribonucleotide asexogenous. In some embodiments, the immunoprotein binding site is anucleotide sequence that binds to an immunoprotein and aids in hidingthe circular polyribonucleotide as exogenous or foreign.

Traditional mechanisms of ribosome engagement to linear RNA involveribosome binding to the capped 5′ end of an RNA. From the 5′ end, theribosome migrates to an initiation codon, whereupon the first peptidebond is formed. According to the present disclosure, internal initiation(i.e., cap-independent) of translation of the circularpolyribonucleotide does not require a free end or a capped end. Rather,a ribosome binds to a non-capped internal site, whereby the ribosomebegins polypeptide elongation at an initiation codon. In someembodiments, the circular polyribonucleotide includes one or more RNAsequences including a ribosome binding site, e.g., an initiation codon.

Natural 5′UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG (SEQ ID NO: 79), where R is a purine (adenine or guanine)three bases upstream of the start codon (AUG), which is followed byanother ‘G’. 5 ‘UTR also have been known to form secondary structureswhich are involved in elongation factor binding.

In some embodiments, a circular polyribonucleotide encodes a proteinbinding sequence that binds to a protein. In some embodiments, theprotein binding sequence targets or localizes the circularpolyribonucleotide to a specific target. In some embodiments, theprotein binding sequence specifically binds an arginine-rich region of aprotein.

In some embodiments, the protein binding site includes, but is notlimited to, a binding site to the protein such as ACIN1, AGO, APOBEC3F,APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7,CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3,EIF4G2, ELAVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1,FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM,HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7,LIN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-,NOP58, NPM1, NUDT21, PCBP2, POLR2A, PRPF8, PTBP1, RBFOX2, RBM10, RBM22,RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM,SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1,TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1,YTHDC1, YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1, and any other proteinthat binds RNA.

Spacer Sequences

In some embodiments, the polyribonucleotide described herein includesone or more spacer sequences. A spacer refers to any contiguousnucleotide sequence (e.g., of one or more nucleotides) that providesdistance or flexibility between two adjacent polynucleotide regions.Spacers may be present in between any of the nucleic acid elementsdescribed herein. Spacers may also be present within a nucleic acidelement described herein.

For example, wherein a nucleic acid includes any two or more of thefollowing elements: (A) a 3′ half of Group I catalytic intron fragment;(B) a 3′ splice site; (C) a 3′ exon fragment; (D) a polyribonucleotidecargo; (E) a 5′ exon fragment; (F) a 5′ splice site; and (G) a 5′ halfof Group I catalytic intron fragment; a spacer region may be presentbetween any one or more of the elements. Any of elements (A), (B), (C),(D), (E), (F), or (G) may be separated by a spacer sequence, asdescribed herein. For example, there may be a spacer between (A) and(B), between (B) and (C), between (C) and (D), between (D) and (E),between (E) and (F), or between (F) and (G).

In some embodiments, the polyribonucleotide further includes a firstspacer region between the 5′ exon fragment of (C) and thepolyribonucleotide cargo of (D). The spacer may be, e.g., at least 5(e.g., at least 10, at least 15, at least 20) ribonucleotides in length.In some embodiments, the polyribonucleotide further includes a secondspacer region between the polyribonucleotide cargo of (D) and the 5′exon fragment of (E). The spacer may be, e.g., at least 5 (e.g., atleast 10, at least 15, at least 20) ribonucleotides in length. In someembodiments, each spacer region is at least 5 (e.g., at least 10, atleast 15, at least 20) ribonucleotides in length. Each spacer region maybe, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in length.The first spacer region, the second spacer region, or the first spacerregion and the second spacer region may include a polyA sequence. Thefirst spacer region, the second spacer region, or the first spacerregion and the second spacer region may include a polyA-C sequence. Insome embodiments, the first spacer region, the second spacer region, orthe first spacer region and the second spacer region includes a polyA-Gsequence. In some embodiments, the first spacer region, the secondspacer region, or the first spacer region and the second spacer regionincludes a polyA-T sequence. In some embodiments, the first spacerregion, the second spacer region, or the first spacer region and thesecond spacer region includes a random sequence.

Spacers may also be present within a nucleic acid region describedherein. For example, a polynucleotide cargo region may include one ormultiple spacers. Spacers may separate regions within the polynucleotidecargo.

In some embodiments, the spacer sequence can be, for example, at least10 nucleotides in length, at least 15 nucleotides in length, or at least30 nucleotides in length. In some embodiments, the spacer sequence is atleast 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30nucleotides in length. In some embodiments, the spacer sequence is nomore than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides inlength. In some embodiments the spacer sequence is from 20 to 50nucleotides in length. In certain embodiments, the spacer sequence is10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides in length.

The spacer sequences can be polyA sequences, polyA-C sequences, polyCsequences, or poly-U sequences.

In some embodiments, the spacer sequences can be polyA-T, polyA-C,polyA-G, or a random sequence.

A spacer sequences may be used to separate an IRES from adjacentstructural elements to martini the structure and function of the IRES orthe adjacent element. A spacer can be specifically engineered dependingon the IRES. In some embodiments, an RNA folding computer software, suchas RNAFold, can be utilized to guide designs of the various elements ofthe vector, including the spacers.

In some embodiments, the polyribonucleotide includes a 5′ spacersequence (e.g., between the 5′ annealing region and thepolyribonucleotide cargo). In some embodiments, the 5′ spacer sequenceis at least 10 nucleotides in length. In another embodiment, the 5′spacer sequence is at least 15 nucleotides in length. In a furtherembodiment, the 5′ spacer sequence is at least 30 nucleotides in length.In some embodiments, the 5′ spacer sequence is at least 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. Insome embodiments, the 5′ spacer sequence is no more than 100, 90, 80,70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodimentsthe 5′ spacer sequence is between 20 and 50 nucleotides in length. Incertain embodiments, the 5′ spacer sequence is 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50nucleotides in length. In one embodiment, the 5′ spacer sequence is apolyA sequence. In another embodiment, the 5′ spacer sequence is apolyA-C sequence. In some embodiments, the 5′ spacer sequence includes apolyA-G sequence. In some embodiments, the 5′ spacer sequence includes apolyA-T sequence. In some embodiments, the 5′ spacer sequence includes arandom sequence.

In some embodiments, the polyribonucleotide includes a 3′ spacersequence (e.g., between the 3′ annealing region and thepolyribonucleotide cargo). In some embodiments, the 3′ spacer sequenceis at least 10 nucleotides in length. In another embodiment, the 3′spacer sequence is at least 15 nucleotides in length. In a furtherembodiment, the 3′ spacer sequence is at least 30 nucleotides in length.In some embodiments, the 3′ spacer sequence is at least 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. Insome embodiments, the 3′ spacer sequence is no more than 100, 90, 80,70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodimentsthe 3′ spacer sequence is from 20 to 50 nucleotides in length. Incertain embodiments, the 3′ spacer sequence is 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50nucleotides in length. In one embodiment, the 3′ spacer sequence is apolyA sequence. In another embodiment, the 5′ spacer sequence is apolyA-C sequence. In some embodiments, the 5′ spacer sequence includes apolyA-G sequence. In some embodiments, the 5′ spacer sequence includes apolyA-T sequence. In some embodiments, the 5′ spacer sequence includes arandom sequence.

In one embodiment, the polyribonucleotide includes a 5′ spacer sequence,but not a 3′ spacer sequence. In another embodiment, thepolyribonucleotide includes a 3′ spacer sequence, but not a 5′ spacersequence. In another embodiment, the polyribonucleotide includes neithera 5′ spacer sequence, nor a 3′ spacer sequence. In another embodiment,the polyribonucleotide does not include an IRES sequence. In a furtherembodiment, the polyribonucleotide does not include an IRES sequence, a5′ spacer sequence or a 3′ spacer sequence.

In some embodiments, the spacer sequence includes at least 3ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides,at least about 8 ribonucleotides, at least about 10 ribonucleotides, atleast about 12 ribonucleotides, at least about 15 ribonucleotides, atleast about 20 ribonucleotides, at least about 25 ribonucleotides, atleast about 30 ribonucleotides, at least about 40 ribonucleotides, atleast about 50 ribonucleotides, at least about 60 ribonucleotides, atleast about 70 ribonucleotides, at least about 80 ribonucleotides, atleast about 90 ribonucleotides, at least about 100 ribonucleotides, atleast about 120 ribonucleotides, at least about 150 ribonucleotides, atleast about 200 ribonucleotides, at least about 250 ribonucleotides, atleast about 300 ribonucleotides, at least about 400 ribonucleotides, atleast about 500 ribonucleotides, at least about 600 ribonucleotides, atleast about 700 ribonucleotides, at least about 800 ribonucleotides, atleast about 900 ribonucleotides, or at least about 100 ribonucleotides.

Methods of Production Methods of Production in a Cell-Free System

The disclosure also provides methods of producing a circular RNA. Forexample, a deoxyribonucleotide template may be transcribed in acell-free system (e.g., by in vitro transcription) to a produce a linearRNA. The linear polyribonucleotide produces a splicing-compatiblepolyribonucleotide, which may be self-spliced to produce a circularpolyribonucleotide.

In some embodiments, the disclosure provides a method of producing acircular polyribonucleotide (e.g., in a cell-free system) by providing alinear polyribonucleotide; and self-splicing linear polyribonucleotideunder conditions suitable for splicing of the 3′ and 5′ splice sites ofthe linear polyribonucleotide; thereby producing a circularpolyribonucleotide.

In some embodiments, the disclosure provides a method of producing acircular polyribonucleotide by providing a deoxyribonucleotide encodingthe linear polyribonucleotide; transcribing the deoxyribonucleotide in acell-free system to produce the linear polyribonucleotide; optionallypurifying the splicing-compatible linear polyribonucleotide; andself-splicing the linear polyribonucleotide under conditions suitablefor splicing of the 3′ and 5′ splice sites of the linearpolyribonucleotide, thereby producing a circular polyribonucleotide.

In some embodiments, the disclosure provides a method of producing acircular polyribonucleotide by providing a deoxyribonucleotide encodinga linear polyribonucleotide; transcribing the deoxyribonucleotide in acell-free system to produce the linear polyribonucleotide, wherein thetranscribing occurs in a solution under conditions suitable for splicingof the 3′ and 5′ splice sites of the linear polyribonucleotide, therebyproducing a circular polyribonucleotide. In some embodiments, the linearpolyribonucleotide comprises a 5′ split-intron and a 3′ split-intron(e.g., a self-splicing construct for producing a circularpolyribonucleotide). In some embodiments, the linear polyribonucleotidecomprises a 5′ annealing region and a 3′ annealing region.

Suitable conditions for in vitro transcriptions and or self-splicing mayinclude any conditions (e.g., a solution or a buffer, such as an aqueousbuffer or solution) that mimic physiological conditions in one or morerespects. In some embodiments, suitable conditions include between0.1-100 mM Mg2+ ions or a salt thereof (e.g., 1-100 mM, 1-50 mM, 1-20mM, 5- 50 mM, 5-20 mM, or 5-15 mM). In some embodiments, suitableconditions include between 1-1000 mM K+ ions or a salt thereof such asKCI (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50- 500 mM, 100-500 mM, or100-300 mM). In some embodiments, suitable conditions include between1-1000 mM Cl- ions or a salt thereof such as KCI (e.g., 1-1000 mM, 1-500mM, 1-200 mM, 50- 500 mM, 100-500 mM, or 100-300 mM). In someembodiments, suitable conditions include between 0.1-100 mM Mn2+ ions ora salt thereof such as MnCI2 (e.g., 0.1-100 mM, 0.1-50 mM, 0.1-20 mM,0.1-10 mM, 0.1-5 mM, 0.1-2 mM, 0.5- 50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5mM, 0.5-2 mM, or 0.1-10 mM). In some embodiments, suitable conditionsinclude dithiothreitol (DTT) (e.g., 1-1000 µM, 1-500 µM, 1-200 µM, 50-500 µM, 100-500 µM, 100-300 µM, 0.1-100 mM, 0.1-50 mM, 0.1-20 mM, 0.1-10mM, 0.1-5 mM, 0.1-2 mM, 0.5- 50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5 mM,0.5-2 mM, or 0.1-10 mM). In some embodiments, suitable conditionsinclude between 0.1 mM and 100 mM ribonucleoside triphosphate (NTP)(e.g., 0.1-100 mM, 0.1-50 mM, 0.1-10 mM, 1- 100 mM, 1-50 mM, or 1-10mM). In some embodiments, suitable conditions include a pH of 4 to 10(e.g., pH of 5 to 9, pH of 6 to 9, or pH of 6.5 to 8.5). In someembodiments, suitable conditions include a temperature of 4° C. to 50°C. (e.g., 10° C. to 40° C., 15° C. to 40° C., 20° C. to 40° C., or 30°C. to 40° C.),

In some embodiments the linear polyribonucleotide is produced from adeoxyribonucleic acid, e.g., a deoxyribonucleic acid described herein,such as a DNA vector, a linearized DNA vector, or a cDNA. In someembodiments, the linear polyribonucleotide is transcribed from thedeoxyribonucleic acid by transcription in a cell-free system (e.g., invitro transcription).

Methods of Production in a Cell

The disclosure also provides methods of producing a circular RNA in acell, e.g., a prokaryotic cell or a eukaryotic cell. In someembodiments, an exogenous polyribonucleotide is provided to a cell(e.g., a linear polyribonucleotide described herein or a DNA moleculeencoding for the transcription of a linear polyribonucleotide describedhere). The linear polyribonucleotides may be transcribed in the cellfrom an exogenous DNA molecule provided to the cell. The linearpolyribonucleotide may be transcribed in the cell from an exogenousrecombinant DNA molecule transiently provided to the cell. In someembodiments, the exogenous DNA molecule does not integrate into thecell’s genome. In some embodiments, the linear polyribonucleotide istranscribed in the cell from a recombinant DNA molecule that isincorporated into the cell’s genome.

In some embodiments, the cell is a prokaryotic cell. In someembodiments, the prokaryotic cell including the polyribonucleotidesdescribed herein may be a bacterial cell or an archaeal cell. Forexample, the prokaryotic cell including the polyribonucleotidesdescribed herein may be E coli, halophilic archaea (e.g., Haloferaxvolcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus,Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces,actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida),Bacillus spp. (e.g., Bacillus subtilis, Bacillus anthracis, Bacilluscereus), betaproteobacteria (e.g., Burkholderia), alphaproteobacterial(e.g., Agrobacterium), Pseudomonas (e.g., Pseudomonas putida), andenterobacteria. The prokaryotic cells may be grown in a culture medium.The prokaryotic cells may be contained in a bioreactor.

In some embodiments, the cell is a eukaryotic cell. In some embodiments,the eukaryotic cell including the polyribonucleotides described hereinis a unicellular eukaryotic cell. In some embodiments, the unicellulareukaryotic is a unicellular fungal cell such as a yeast cell (e.g.,Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomycesspp., Schizosaccharomyces spp., Torulaspora spp, and Pichia spp.). Insome embodiments, the unicellular eukaryotic cell is a unicellularanimal cell. A unicellular animal cell may be a cell isolated from amulticellular animal and grown in culture, or the daughter cellsthereof. In some embodiments, the unicellular animal cell may bededifferentiated. In some embodiments, the unicellular eukaryotic cellis a unicellular plant cell. A unicellular plant cell may be a cellisolated from a multicellular plant and grown in culture, or thedaughter cells thereof. In some embodiments, the unicellular plant cellmay be dedifferentiated. In some embodiments, the unicellular plant cellis from a plant callus. In embodiments, the unicellular cell is a plantcell protoplast. In some embodiments, the unicellular eukaryotic cell isa unicellular eukaryotic algal cell, such as a unicellular green alga, adiatom, a euglenid, or a dinoflagellate. Non-limiting examples ofunicellular eukaryotic algae of interest include Dunaliella salina,Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis,Neochloris oleoabundans and other Neochloris spp., Protosiphonbotryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonasreinhardtii and other Chlamydomonas spp. In some embodiments, theunicellular eukaryotic cell is a protist cell. In some embodiments, theunicellular eukaryotic cell is a protozoan cell.

In some embodiments, the eukaryotic cell is a cell of a multicellulareukaryote. For example, the multicellular eukaryote may be selected fromthe group consisting of a vertebrate animal, an invertebrate animal, amulticellular fungus, a multicellular alga, and a multicellular plant.In some embodiments, the eukaryotic organism is a human. In someembodiments, the eukaryotic organism is a non-human vertebrate animal.In some embodiments, the eukaryotic organism is an invertebrate animal.In some embodiments, the eukaryotic organism is a multicellular fungus.In some embodiments, the eukaryotic organism is a multicellular plant.In embodiments, the eukaryotic cell is a cell of a human or a cell of anon-human mammal such as a non-human primate (e.g., monkeys, apes),ungulate (e.g., bovids including cattle, buffalo, bison, sheep, goat,and musk ox; pig; camelids including camel, llama, and alpaca; deer,antelope; and equids including horse and donkey), carnivore (e.g., dog,cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), orlagomorph (e.g., rabbit, hare). In embodiments, the eukaryotic cell is acell of a bird, such as a member of the avian taxa Galliformes (e.g.,chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese),Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons,doves), or Psittaciformes (e.g., parrots). In embodiments, theeukaryotic cell is a cell of an arthropod (e.g., insects, arachnids,crustaceans), a nematode, an annelid, a helminth, or a mollusc. Inembodiments, the eukaryotic cell is a cell of a multicellular plant,such as an angiosperm plant (which can be a dicot or a monocot) or agymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), afern, horsetail, clubmoss, or a bryophyte. In embodiments, theeukaryotic cell is a cell of a eukaryotic multicellular alga.

The eukaryotic cells may be grown in a culture medium. The eukaryoticcells may be contained in a bioreactor.

Methods of Purification

One or more purification steps may be included in the methods describedherein. For example, in some embodiments, the linear polyribonucleotideis substantively enriched or pure (e.g., purified) prior toself-splicing the linear polyribonucleotide. In other embodiments, thelinear polyribonucleotide is not purified prior to self-splicing thelinear polyribonucleotide. In some embodiments, the resulting circularRNA is purified.

Purification may include separating or enriching the desired reactionproduct from one or more undesired components, such as any unreactedstating material, byproducts, enzymes, or other reaction components. Forexample, purification of linear polyribonucleotide followingtranscription in a cell-free system (e.g., in vitro transcription) mayinclude separation or enrichment from the DNA template prior toself-splicing the linear polyribonucleotide. Purification of thecircular RNA product following splicing may be used to separate orenrich the circular RNA from its corresponding linear RNA. Methods ofpurification of RNA are known to those of skill in the art and includeenzymatic purification or by chromatography.

In some embodiments, the methods of purification result in a circularpolyribonucleotide that has less than 50% (e.g., less than 40%, 30%,20%, 10%, 5%, 4%, 3%, 2%, or 1%) linear polyribonucleotides.

Bioreactors

In some embodiments, any method of producing a circularpolyribonucleotide described herein may be performed in a bioreactor. Abioreactor refers to any vessel in which a chemical or biologicalprocess is carried out which involves organisms or biochemically activesubstances derived from such organisms. Bioreactors may be compatiblewith the cell-free methods for production of circular RNA describedherein. A vessel for a bioreactor may include a culture flask, a dish,or a bag that may be single use (disposable), autoclavable, orsterilizable. A bioreactor may be made of glass, or it may bepolymer-based, or it may be made of other materials.

Examples of bioreactors include, without limitation, stirred tank (e.g.,well mixed) bioreactors and tubular (e.g., plug flow) bioreactors,airlift bioreactors, membrane stirred tanks, spin filter stirred tanks,vibromixers, fluidized bed reactors, and membrane bioreactors. The modeof operating the bioreactor may be a batch or continuous processes. Abioreactor is continuous when the reagent and product streams arecontinuously being fed and withdrawn from the system. A batch bioreactormay have a continuous recirculating flow, but no continuous feeding ofreagents or product harvest.

Some methods of the present disclosure are directed to large-scaleproduction of circular polyribonucleotides. For large-scale productionmethods, the method may be performed in a volume of 1 liter (L) to 50 L,or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50L, or more). In some embodiments, the method may be performed in avolume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 Lto 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to40 L, 15 L to 45 L, or 15 to 50 L.

In some embodiments, a bioreactor may produce at least 1 g of circularRNA. In some embodiments, a bioreactor may produce 1-200 g of circularRNA (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, of50-200 g of circular RNA). In some embodiments, the amount produced ismeasured per liter (e.g., 1-200 g per liter), per batch or reaction(e.g., 1-200 g per batch or reaction), or per unit time (e.g., 1-200 gper hour or per day).

In some embodiments, more than one bioreactor may be utilized in seriesto increase the production capacity (e.g., one, two, three, four, five,six, seven, eight, or nine bioreactors may be used in series).

Methods of Use

In some embodiments, circular polyribonucleotides made as describedherein are used as effectors in therapy or agriculture.

For example, a circular polyribonucleotide made by the methods describedherein may be administered to a subject (e.g., in a pharmaceutical,veterinary, or agricultural composition). In some embodiments, thesubject is a vertebrate animal (e.g., mammal, bird, fish, reptile, oramphibian). In some embodiments, the subject is a human. In someembodiments, the subject is a non-human mammal. In embodiments, thesubject is a non-human mammal is such as a non-human primate (e.g.,monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig,camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog,cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). Inembodiments, the subject is a bird, such as a member of the avian taxaGalliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes(e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus),Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).In embodiments, the subject is an invertebrate such as an arthropod(e.g., insects, arachnids, crustaceans), a nematode, an annelid, ahelminth, or a mollusk. In embodiments, the subject is an invertebrateagricultural pest or an invertebrate that is parasitic on aninvertebrate or vertebrate host. In embodiments, the subject is a plant,such as an angiosperm plant (which can be a dicot or a monocot) or agymnosperm plant (e.g., a conifer, a cycad, a gnetophyte, a Ginkgo), afern, horsetail, clubmoss, or a bryophyte. In embodiments, the subjectis a eukaryotic alga (unicellular or multicellular). In embodiments, thesubject is a plant of agricultural or horticultural importance, such asrow crop plants, fruit-producing plants and trees, vegetables, trees,and ornamental plants including ornamental flowers, shrubs, trees,groundcovers, and turf grasses.

In some embodiments, the disclosure provides a method of modifying asubject by providing to the subject a composition or formulationdescribed herein. In some embodiments, the composition or formulation isor includes a nucleic acid molecule (e.g., a DNA molecule or an RNAmolecule described herein), and the polynucleotide is provided to aeukaryotic subject. In some embodiments, the composition or formulationis or includes or a eukaryotic or prokaryotic cell including a nucleicacid described herein.

In some embodiments, the disclosure provides a method of treating acondition in a subject in need thereof by providing to the subject acomposition or formulation described herein. In some embodiments, thecomposition or formulation is or includes a nucleic acid molecule (e.g.,a DNA molecule or an RNA molecule described herein), and thepolynucleotide is provided to a eukaryotic subject. In some embodiments,the composition or formulation is or includes a eukaryotic orprokaryotic cell including a nucleic acid described herein.

In some embodiments, the disclosure provides a method of providing acircular polyribonucleotide to a subject by providing a eukaryotic orprokaryotic cell include a polynucleotide described herein to thesubject.

Formulations

In some embodiments of the present disclosure a circularpolyribonucleotide described herein may be formulated in composition,e.g., a composition for delivery to a cell, a plant, an invertebrateanimal, a non-human vertebrate animal, or a human subject, e.g., anagricultural, veterinary, or pharmaceutical composition. In someembodiments, the circular polyribonucleotide is formulated in apharmaceutical composition. In some embodiments, a composition includesa circular polyribonucleotide and a diluent, a carrier, an adjuvant, ora combination thereof. In a particular embodiment, a compositionincludes a circular polyribonucleotide described herein and a carrier ora diluent free of any carrier. In some embodiments, a compositionincluding a circular polyribonucleotide with a diluent free of anycarrier is used for naked delivery of the circular polyribonucleotide toa subject.

Salts

In some cases, a composition or pharmaceutical composition providedherein comprises one or more salts. For controlling the tonicity, aphysiological salt such as sodium salt can be included a compositionprovided herein. Other salts can comprise potassium chloride, potassiumdihydrogen phosphate, disodium phosphate, and/or magnesium chloride, orthe like. In some cases, the composition is formulated with one or morepharmaceutically acceptable salts. The one or more pharmaceuticallyacceptable salts can comprise those of the inorganic ions, such as, forexample, sodium, potassium, calcium, magnesium ions, and the like. Suchsalts can comprise salts with inorganic or organic acids, such ashydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid,sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, aceticacid, fumaric acid, succinic acid, lactic acid, mandelic acid, malicacid, citric acid, tartaric acid, or maleic acid. The polyribonucleotidecan be present in either linear or circular form.

Buffers/pH

A composition or pharmaceutical composition provided herein can compriseone or more buffers, such as a Tris buffer; a borate buffer; a succinatebuffer; a histidine buffer (e.g., with an aluminum hydroxide adjuvant);or a citrate buffer. Buffers, in some cases, are included in the 5-20 mMrange.

A composition or pharmaceutical composition provided herein can have apH between about 5.0 and about 8.5, between about 6.0 and about 8.0,between about 6.5 and about 7.5, or between about 7.0 and about 7.8. Thecomposition or pharmaceutical composition can have a pH of about 7. Thepolyribonucleotide can be present in either linear or circular form.

Detergents/Surfactants

A composition or pharmaceutical composition provided herein can compriseone or more detergents and/or surfactants, depending on the intendedadministration route, e.g., polyoxyethylene sorbitan esters surfactants(commonly referred to as “Tweens”), e.g., polysorbate 20 and polysorbate80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/orbutylene oxide (BO), sold under the DOWFAX™ tradename, such as linearEO/PO block copolymers; octoxynols, which can vary in the number ofrepeating ethoxy (oxy-1,2-ethanediyl) groups, e.g., octoxynol-9 (TritonX-100, or t-octylphenoxypolyethoxyethanol);(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as “SPANs”), such as sorbitan trioleate (Span 85)and sorbitan monolaurate, an octoxynol (such as octoxynol-9 (TritonX-100) or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammoniumbromide (“CTAB”), or sodium deoxycholate. The one or more detergentsand/or surfactants can be present only at trace amounts. In some cases,the composition can include less than 1 mg/ml of each of octoxynol-10and polysorbate 80. Non-ionic surfactants can be used herein.Surfactants can be classified by their “HLB” (hydrophile/lipophilebalance). In some cases, surfactants have a HLB of at least 10, at least15, and/or at least 16. The polyribonucleotide can be present in eitherlinear or circular form.

Diluents

In some embodiments, a composition of the disclosure includes a circularpolyribonucleotide and a diluent. In some embodiments, a composition ofthe disclosure includes a linear polyribonucleotide and a diluent.

A diluent can be a non-carrier excipient. A non-carrier excipient servesas a vehicle or medium for a composition, such as a circularpolyribonucleotide as described herein. A non-carrier excipient servesas a vehicle or medium for a composition, such as a linearpolyribonucleotide as described herein. Non-limiting examples of anon-carrier excipient include solvents, aqueous solvents, non-aqueoussolvents, dispersion media, diluents, dispersions, suspension aids,surface active agents, isotonic agents, thickening agents, emulsifyingagents, preservatives, polymers, peptides, proteins, cells,hyaluronidases, dispersing agents, granulating agents, disintegratingagents, binding agents, buffering agents (e.g., phosphate bufferedsaline (PBS)), lubricating agents, oils, and mixtures thereof. Anon-carrier excipient can be any one of the inactive ingredientsapproved by the United States Food and Drug Administration (FDA) andlisted in the Inactive Ingredient Database that does not exhibit acell-penetrating effect. A non-carrier excipient can be any inactiveingredient suitable for administration to a non-human animal, forexample, suitable for veterinary use. Modification of compositionssuitable for administration to humans in order to render thecompositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation.

In some embodiments, the circular polyribonucleotide may be delivered asa naked delivery formulation, such as including a diluent. A nakeddelivery formulation delivers a circular polyribonucleotide, to a cellwithout the aid of a carrier and without modification or partial orcomplete encapsulation of the circular polyribonucleotide, cappedpolyribonucleotide, or complex thereof.

A naked delivery formulation is a formulation that is free from acarrier and wherein the circular polyribonucleotide is without acovalent modification that binds a moiety that aids in delivery to acell or without partial or complete encapsulation of the circularpolyribonucleotide. In some embodiments, a circular polyribonucleotidewithout a covalent modification that binds a moiety that aids indelivery to a cell is a polyribonucleotide that is not covalently boundto a protein, small molecule, a particle, a polymer, or a biopolymer. Acircular polyribonucleotide without covalent modification that binds amoiety that aids in delivery to a cell does not contain a modifiedphosphate group. For example, a circular polyribonucleotide without acovalent modification that binds a moiety that aids in delivery to acell does not contain phosphorothioate, phosphoroselenates,boranophosphates, boranophosphate esters, hydrogen phosphonates,phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, orphosphotriesters.

In some embodiments, a naked delivery formulation is free of any or allof: transfection reagents, cationic carriers, carbohydrate carriers,nanoparticle carriers, or protein carriers. In some embodiments, a nakeddelivery formulation is free from phtoglycogen octenyl succinate,phytoglycogen beta-dextrin, anhydride-modified phytoglycogenbeta-dextrin, lipofectamine, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3- Trimethylammonium-Propane(DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA),3B-[N-(N\N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl), diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),human serum albumin (HSA), low-density lipoprotein (LDL), high-densitylipoprotein (HDL), or globulin.

In certain embodiments, a naked delivery formulation includes anon-carrier excipient. In some embodiments, a non-carrier excipientincludes an inactive ingredient that does not exhibit a cell-penetratingeffect. In some embodiments, a non-carrier excipient includes a buffer,for example PBS. In some embodiments, a non-carrier excipient is asolvent, a non-aqueous solvent, a diluent, a suspension aid, asurface-active agent, an isotonic agent, a thickening agent, anemulsifying agent, a preservative, a polymer, a peptide, a protein, acell, a hyaluronidase, a dispersing agent, a granulating agent, adisintegrating agent, a binding agent, a buffering agent, a lubricatingagent, or an oil.

In some embodiments, a naked delivery formulation includes a diluent. Adiluent may be a liquid diluent or a solid diluent. In some embodiments,a diluent is an RNA solubilizing agent, a buffer, or an isotonic agent.Examples of an RNA solubilizing agent include water, ethanol, methanol,acetone, formamide, and 2-propanol. Examples of a buffer include2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris,2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 3-(N-morpholino)propanesulfonic acid (MOPS),4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris,Tricine, Gly-Gly, Bicine, or phosphate. Examples of an isotonic agentinclude glycerin, mannitol, polyethylene glycol, propylene glycol,trehalose, or sucrose.

Carriers

In some embodiments, a composition of the disclosure includes a circularpolyribonucleotide and a carrier. In some embodiments, a composition ofthe disclosure includes a linear polyribonucleotide and a carrier.

In certain embodiments, a composition includes a circularpolyribonucleotide as described herein in a vesicle or othermembrane-based carrier. In certain embodiments, a composition includes alinear polyribonucleotide as described herein in a vesicle or othermembrane-based carrier.

In other embodiments, a composition includes the circularpolyribonucleotide in or via a cell, vesicle or other membrane-basedcarrier. In other embodiments, a composition includes the linearpolyribonucleotide in or via a cell, vesicle or other membrane-basedcarrier. In one embodiment, a composition includes the circularpolyribonucleotide in liposomes or other similar vesicles. In oneembodiment, a composition includes the linear polyribonucleotide inliposomes or other similar vesicles. Liposomes are spherical vesiclestructures composed of a uni- or multilamellar lipid bilayer surroundinginternal aqueous compartments and a relatively impermeable outerlipophilic phospholipid bilayer. Liposomes may be anionic, neutral, orcationic. Liposomes are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Vesicles can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Methods for preparation of multilamellar vesicle lipids areknown in the art (see for example U.S. Pat. No. 6,693,086, the teachingsof which relating to multilamellar vesicle lipid preparation areincorporated herein by reference). Although vesicle formation can bespontaneous when a lipid film is mixed with an aqueous solution, it canalso be expedited by applying force in the form of shaking by using ahomogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch andNavarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review). Extruded lipids can beprepared by extruding through filters of decreasing size, as describedin Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings ofwhich relating to extruded lipid preparation are incorporated herein byreference.

In certain embodiments, a composition of the disclosure includes acircular polyribonucleotide and lipid nanoparticles, for example lipidnanoparticles described herein. In certain embodiments, a composition ofthe disclosure includes a linear polyribonucleotide and lipidnanoparticles. Lipid nanoparticles are another example of a carrier thatprovides a biocompatible and biodegradable delivery system for acircular polyribonucleotide molecule as described herein. Lipidnanoparticles are another example of a carrier that provides abiocompatible and biodegradable delivery system for a linearpolyribonucleotide molecule as described herein. Nanostructured lipidcarriers (NLCs) are modified solid lipid nanoparticles (SLNs) thatretain the characteristics of the SLN, improve drug stability andloading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs)are an important component of drug delivery. These nanoparticles caneffectively direct drug delivery to specific targets and improve drugstability and controlled drug release. Lipid-polymer nanoparticles(PLNs), a new type of carrier that combines liposomes and polymers, mayalso be employed. These nanoparticles possess the complementaryadvantages of PNPs and liposomes. A PLN is composed of a core-shellstructure; the polymer core provides a stable structure, and thephospholipid shell offers good biocompatibility. As such, the twocomponents increase the drug encapsulation efficiency rate, facilitatesurface modification, and prevent leakage of water-soluble drugs. For areview, see, e.g., Li et al. 2017, Nanomaterials 7, 122;doi:10.3390/nano7060122.

Additional non-limiting examples of carriers include carbohydratecarriers (e.g., an anhydride-modified phytoglycogen or glycogen-typematerial), protein carriers (e.g., a protein covalently linked to thecircular polyribonucleotide or a protein covalently linked to the linearpolyribonucleotide), or cationic carriers (e.g., a cationic lipopolymeror transfection reagent). Non-limiting examples of carbohydrate carriersinclude phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, andanhydride-modified phytoglycogen beta-dextrin. Non-limiting examples ofcationic carriers include lipofectamine, polyethylenimine,poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine,aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine,spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine),poly(histidine), poly(arginine), cationized gelatin, dendrimers,chitosan, 1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA),3B-[N-(N\N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl), diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC).Non-limiting examples of protein carriers include human serum albumin(HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), orglobulin.

Exosomes can also be used as drug delivery vehicles for a circular RNAcomposition or preparation described herein. Exosomes can be used asdrug delivery vehicles for a linear polyribonucleotide composition orpreparation described herein. For a review, see Ha et al. July 2016.Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296;https://doi.org/10.1016/j.apsb.2016.02.001.

Ex vivo differentiated red blood cells can also be used as a carrier fora circular RNA composition or preparation described herein. Ex vivodifferentiated red blood cells can also be used as a carrier for alinear polyribonucleotide composition or preparation described herein.See, e.g., International Patent Publication Nos. WO2015/073587;WO2017/123646; WO2017/123644; WO2018/102740; WO2016/183482;WO2015/153102; WO2018/151829; WO2018/009838; Shi et al. 2014. Proc NatlAcad Sci USA. 111(28): 10131-10136; U.S. Pat. 9,644,180; Huang et al.2017. Nature Communications 8: 423; Shi et al. 2014. Proc Natl Acad SciUSA. 111(28): 10131-10136.

Fusosome compositions, e.g., as described in International PatentPublication No. WO2018/208728, can also be used as carriers to deliver acircular polyribonucleotide molecule described herein. Fusosomecompositions, e.g., as described in WO2018/208728, can also be used ascarriers to deliver a linear polyribonucleotide molecule describedherein.

Virosomes and virus-like particles (VLPs) can also be used as carriersto deliver a circular polyribonucleotide molecule described herein totargeted cells. Virosomes and virus-like particles (VLPs) can also beused as carriers to deliver a linear polyribonucleotide moleculedescribed herein to targeted cells.

Plant nanovesicles and plant messenger packs (PMPs), e.g., as describedin International Patent Publication Nos. WO2011/097480, WO2013/070324,WO2017/004526, or WO2020/041784 can also be used as carriers to deliverthe circular RNA composition or preparation described herein. Plantnanovesicles and plant messenger packs (PMPs) can also be used ascarriers to deliver a linear polyribonucleotide composition orpreparation described herein.

Microbubbles can also be used as carriers to deliver a circularpolyribonucleotide molecule described herein. Microbubbles can also beused as carriers to deliver a linear polyribonucleotide moleculedescribed herein. See, e.g., US7115583; Beeri, R. et al., Circulation.2002 Oct 1;106(14):1756-1759; Bez, M. et al., Nat Protoc. 2019 Apr;14(4): 1015-1026; Hernot, S. et al., Adv Drug Deliv Rev. 2008 Jun 30;60(10): 1153-1166; Rychak, J.J. et al., Adv Drug Deliv Rev. 2014 Jun;72: 82-93. In some embodiments, microbubbles are albumin-coatedperfluorocarbon microbubbles.

The carrier including the circular polyribonucleotides described hereinmay include a plurality of particles. The particles may have medianarticle size of 30 to 700 nanometers (e.g., 30 to 50, 50 to 100, 100 to200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 100 to500, 50 to 500, or 200 to 700 nanometers). The size of the particle maybe optimized to favor deposition of the payload, including the circularpolyribonucleotide into a cell. Deposition of the circularpolyribonucleotide into certain cell types may favor different particlesizes. For example, the particle size may be optimized for deposition ofthe circular polyribonucleotide into antigen presenting cells. Theparticle size may be optimized for deposition of the circularpolyribonucleotide into dendritic cells. Additionally, the particle sizemay be optimized for depositions of the circular polyribonucleotide intodraining lymph node cells.

Lipid Nanoparticles

The compositions, methods, and delivery systems provided by the presentdisclosure may employ any suitable carrier or delivery modalitydescribed herein, including, in certain embodiments, lipid nanoparticles(LNPs). Lipid nanoparticles, in some embodiments, comprise one or moreionic lipids, such as non-cationic lipids (e.g., neutral or anionic, orzwitterionic lipids); one or more conjugated lipids (such asPEG-conjugated lipids or lipids conjugated to polymers described inTable 5 of WO2019217941; incorporated herein by reference in itsentirety); one or more sterols (e.g., cholesterol).

Lipids that can be used in nanoparticle formations (e.g., lipidnanoparticles) include, for example those described in Table 4 ofWO2019217941, which is incorporated by reference—e.g., alipid-containing nanoparticle can comprise one or more of the lipids inTable 4 of WO2019217941. Lipid nanoparticles can include additionalelements, such as polymers, such as the polymers described in Table 5 ofWO2019217941, incorporated by reference.

In some embodiments, conjugated lipids, when present, can include one ormore of PEG-diacylglycerol (DAG) (such asl-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)),PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), apegylated phosphatidylethanoloamine (PEG-PE), PEG succinatediacylglycerol (PEGS-DAG) (such as4-0-(2′,3′-di(tetradecanoyioxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam,N-(carbonyl-methoxypoly ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and thosedescribed in Table 2 of WO2019051289 (incorporated by reference), andcombinations of the foregoing.

In some embodiments, sterols that can be incorporated into lipidnanoparticles include one or more of cholesterol or cholesterolderivatives, such as those in W02009/127060 or US2010/0130588, which areincorporated by reference. Additional exemplary sterols includephytosterols, including those described in Eygeris et al. (2020),dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein byreference.

In some embodiments, the lipid particle comprises an ionizable lipid, anon-cationic lipid, a conjugated lipid that inhibits aggregation ofparticles, and a sterol. The amounts of these components can be variedindependently and to achieve desired properties. For example, in someembodiments, the lipid nanoparticle comprises an ionizable lipid is inan amount from about 20 mol% to about 90 mol% of the total lipids (inother embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol);about 50 mol% to about 90 mol% of the total lipid present in the lipidnanoparticle), a non-cationic lipid in an amount from about 5 mol% toabout 30 mol% of the total lipids, a conjugated lipid in an amount fromabout 0.5 mol% to about 20 mol% of the total lipids, and a sterol in anamount from about 20 mol% to about 50 mol% of the total lipids. Theratio of total lipid to nucleic acid can be varied as desired. Forexample, the total lipid to nucleic acid (mass or weight) ratio can befrom about 10: 1 to about 30: 1.

In some embodiments, the lipid to nucleic acid ratio (mass/mass ratio;w/w ratio) can be in the range of from about 1:1 to about 25:1, fromabout 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.The amounts of lipids and nucleic acid can be adjusted to provide adesired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 orhigher. Generally, the lipid nanoparticle formulation’s overall lipidcontent can range from about 5 mg/ml to about 30 mg/mL.

Some non-limiting example of lipid compounds that may be used (e.g., incombination with other lipid components) to form lipid nanoparticles forthe delivery of compositions described herein, e.g., nucleic acid (e.g.,RNA (e.g., circular polyribonucleotide, linear polyribonucleotide))described herein includes,

In some embodiments an LNP comprising Formula (i) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising Formula (ii) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising Formula (iii) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising Formula (v) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising Formula (vi) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising Formula (viii) is used to delivera polyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising Formula (ix) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

wherein X¹ is O, NR¹, or a direct bond, X² is C2-5 alkylene, X³ is C(═O)or a direct bond, R¹ is H or Me, R³ is C1-3 alkyl, R² is C1-3 alkyl, orR² taken together with the nitrogen atom to which it is attached and 1-3carbon atoms of X² form a 4-, 5-, or 6-membered ring, or X¹ is NR¹, R¹and R² taken together with the nitrogen atoms to which they are attachedform a 5- or 6-membered ring, or R² taken together with R³ and thenitrogen atom to which they are attached form a 5-, 6-, or 7-memberedring, Y¹ is C2-12 alkylene, Y² is selected from

(in either orientation), (in either orientation), (in eitherorientation), n is 0 to 3, R⁴ is C1-15 alkyl, Z¹ is C1-6 alkylene or adirect bond, Z² is

(in either orientation) or absent, provided that if Z¹ is a direct bond,Z² is absent; R⁵ is C5-9 alkyl or C6-10 alkoxy, R⁶ is C5-9 alkyl orC6-10 alkoxy, W is methylene or a direct bond, and R⁷ is H or Me, or asalt thereof, provided that if R³ and R² are C2 alkyls, X¹ is O, X² islinear C3 alkylene, X³ is C(=0), Y¹ is linear Ce alkylene, (Y²)n-R⁴ is

, R⁴ is linear C5 alkyl, Z¹ is C2 alkylene, Z² is absent, W ismethylene, and R⁷ is H, then R⁵ and R⁶ are not Cx alkoxy.

In some embodiments an LNP comprising Formula (xii) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising Formula (xi) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprises a compound of Formula (xiii) and acompound of Formula (xiv).

In some embodiments an LNP comprising Formula (xv) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells.

In some embodiments an LNP comprising a formulation of Formula (xvi) isused to deliver a polyribonucleotide (e.g., a circularpolyribonucleotide, a linear polyribonucleotide) composition describedherein to cells.

In some embodiments, a lipid compound used to form lipid nanoparticlesfor the delivery of compositions described herein, e.g., nucleic acid(e.g., RNA (e.g., circular polyribonucleotide, linearpolyribonucleotide)) described herein is made by one of the followingreactions:

In some embodiments an LNP comprising Formula (xxi) is used to deliver apolyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells. In someembodiments the LNP of Formula (xxi) is an LNP described by WO2021113777(e.g., a lipid of Formula (1) such as a lipid of Table 1 ofWO2021113777).

wherein

-   each n is independently an integer from 2-15; L₁ and L₃ are each    independently —OC(O)—* or —C(O)O—*, wherein “*” indicates the    attachment point to R₁ or R₃;

-   R₁ and R₃ are each independently a linear or branched C₉-C₂₀ alkyl    or C₉-C₂₀ alkenyl, optionally substituted by one or more    substituents selected from a group consisting of oxo, halo, hydroxy,    cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl,    dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl,    dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl,    heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino,    aminoalkylcarbonylamino, aminocarbonylalkylamino,    (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino,    hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl,    aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl,    dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,    (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl,    dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl,    alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkyl    sulfonyl, and alkyl sulfonealkyl; and

-   R₂ is selected from a group consisting of:

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

In some embodiments an LNP comprising Formula (xxii) is used to delivera polyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells. In someembodiments the LNP of Formula (xxii) is an LNP described byWO2021113777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 ofWO2021113777).

wherein

-   each n is independently an integer from 1-15;

-   R₁ and R₂ are each independently selected from a group consisting    of:

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   R₃ is selected from a group consisting of:

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

In some embodiments an LNP comprising Formula (xxiii) is used to delivera polyribonucleotide (e.g., a circular polyribonucleotide, a linearpolyribonucleotide) composition described herein to cells. In someembodiments the LNP of Formula (xxiii) is an LNP described byWO2021113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 ofWO2021113777).

wherein

-   X is selected from —O—, —S—, or —OC(O)—*, wherein * indicates the    attachment point to R₁;

-   R₁ is selected from a group consisting of:

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   and R₂ is selected from a group consisting of:

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

-   

In some embodiments, a composition described herein (e.g., a nucleicacid (e.g., a circular polyribonucleotide, a linear polyribonucleotide)or a protein) is provided in an LNP that comprises an ionizable lipid.In some embodiments, the ionizable lipid is heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102);e.g., as described in Example 1 of US9,867,888 (incorporated byreference herein in its entirety). In some embodiments, the ionizablelipid is9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate (LP01), e.g., as synthesized in Example 13 ofWO2015/095340 (incorporated by reference herein in its entirety). Insome embodiments, the ionizable lipid is Di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., assynthesized in Example 7, 8, or 9 of US2012/0027803 (incorporated byreference herein in its entirety). In some embodiments, the ionizablelipid is1,1′-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200),e.g., as synthesized in Examples 14 and 16 of WO2010/053572(incorporated by reference herein in its entirety). In some embodiments,the ionizable lipid is Imidazole cholesterol ester (ICE) lipid (3S, 10R,13R, 17R)-10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8,9, 10, 11, 12, 13, 14, 15, 16,17-tetradecahydro-IH-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946(incorporated by reference herein in its entirety).

In some embodiments, an ionizable lipid may be a cationic lipid, anionizable cationic lipid, e.g., a cationic lipid that can exist in apositively charged or neutral form depending on pH, or anamine-containing lipid that can be readily protonated. In someembodiments, the cationic lipid is a lipid capable of being positivelycharged, e.g., under physiological conditions. Exemplary cationic lipidsinclude one or more amine group(s) which bear the positive charge. Insome embodiments, the lipid particle comprises a cationic lipid informulation with one or more of neutral lipids, ionizableamine-containing lipids, biodegradable alkyne lipids, steroids,phospholipids including polyunsaturated lipids, structural lipids (e.g.,sterols), PEG, cholesterol, and polymer conjugated lipids. In someembodiments, the cationic lipid may be an ionizable cationic lipid. Anexemplary cationic lipid as disclosed herein may have an effective pKaover 6.0. In embodiments, a lipid nanoparticle may comprise a secondcationic lipid having a different effective pKa (e.g., greater than thefirst effective pKa), than the first cationic lipid. A lipidnanoparticle may comprise between 40 and 60 mol percent of a cationiclipid, a neutral lipid, a steroid, a polymer conjugated lipid, and atherapeutic agent, e.g., a nucleic acid (e.g., RNA (e.g., a circularpolyribonucleotide, a linear polyribonucleotide)) described herein,encapsulated within or associated with the lipid nanoparticle. In someembodiments, the nucleic acid is co-formulated with the cationic lipid.The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNPcomprising a cationic lipid. In some embodiments, the nucleic acid maybe encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. Insome embodiments, the lipid nanoparticle may comprise a targetingmoiety, e.g., coated with a targeting agent. In embodiments, the LNPformulation is biodegradable. In some embodiments, a lipid nanoparticlecomprising one or more lipid described herein, e.g., Formula (i), (ii),(ii), (vii) and/or (ix) encapsulates at least 1%, at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 92%, at least95%, at least 97%, at least 98% or 100% of an RNA molecule.

Exemplary ionizable lipids that can be used in lipid nanoparticleformulations include, without limitation, those listed in Table 1 ofWO2019051289, incorporated herein by reference. Additional exemplarylipids include, without limitation, one or more of the followingformulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224;I, II or III of US20160151284; I, IA, II, or IIAof US20170210967; I-c ofUS20150140070; A of US2013/0178541 ; I of US2013/0303587 orUS2013/0123338; I of US2015/0141678; II, III, IV, or V ofUS2015/0239926; I of US2017/0119904; I or II of WO2017/117528; A ofUS2012/0149894; A of US2015/0057373; A of WO2013/116126; A ofUS2013/0090372; A of US2013/0274523; A of US2013/0274504; A ofUS2013/0053572; A of W02013/016058; A of W02012/162210; I ofUS2008/042973; I, II, III, or IV of US2012/01287670; I or II ofUS2014/0200257; I, II, or III of US2015/0203446; I or III ofUS2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIVof US2014/0308304; of US2013/0338210; I, II, III, or IV ofW02009/132131; A of US2012/01011478; I or XXXV of US2012/0027796; XIV orXVII of US2012/0058144; of US2013/0323269; I of US2011/0117125; I, II,or III of US2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII of US2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII,XIV, XV, or XVI of US2011/0076335; I or II of US2006/008378; I ofUS2013/0123338; I or X-A-Y-Z of US2015/0064242; XVI, XVII, or XVIII ofUS2013/0022649; I, II, or III of US2013/0116307; I, II, or III ofUS2013/0116307; I or II of US2010/0062967; I-X of US2013/0189351; I ofUS2014/0039032; V of US2018/0028664; I of US2016/0317458; I ofUS2013/0195920; 5, 6, or 10 of US10,221,127; III-3 of WO2018/081480; I-5or I-8 of WO2020/081938; 18 or 25 of US9,867,888; A of US2019/0136231 ;II of WO2020/219876; 1 of US2012/0027803; OF-02 of US2019/0240349; 23 ofUS10,086,013; cKK-E12/A6 of Miao et al (2020); C12-200 of WO2010/053572;7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al;TS-P4C2 of US9,708,628; I of WO2020/106946; I of WO2020/106946; and (1),(2), (3), or (4) of WO2021/113777. Exemplary lipids further include alipid of any one of Tables 1-16 of WO2021/113777.

In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3IZ)-heptatriaconta- 6,9,28,3 I-tetraen-l9-yl-4-(dimethylamino) butanoate(DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9(incorporated by reference herein in its entirety). In some embodiments,the ionizable lipid is the lipid ATX-002, e.g., as described in Example10 of WO2019051289A9 (incorporated by reference herein in its entirety).In some embodiments, the ionizable lipid is(l3Z,l6Z)-A,A-dimethyl-3-nonyldocosa-l3, l6-dien-l-amine (Compound 32),e.g., as described in Example 11 of WO2019051289A9 (incorporated byreference herein in its entirety). In some embodiments, the ionizablelipid is Compound 6 or Compound 22, e.g., as described in Example 12 ofWO2019051289A9 (incorporated by reference herein in its entirety).

Exemplary non-cationic lipids include, but are not limited to,distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE),dimethylphosphatidylethanolamine (such as 16-O-dimethyl PE), l8-l-transPE, l-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), hydrogenated soyphosphatidylcholine (HSPC), egg phosphatidylcholine (EPC),dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG),distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine(DEPC), palmitoyloleyolphosphatidylglycerol (POPG),dielaidoyl-phosphatidylethanolamine (DEPE), lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, eggsphingomyelin (ESM), cephalin, cardiolipin,phosphatidicacid,cerebrosides, dicetylphosphate,lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixturesthereof. It is understood that other diacylphosphatidylcholine anddiacylphosphatidylethanolamine phospholipids can also be used. The acylgroups in these lipids are preferably acyl groups derived from fattyacids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl,stearoyl, or oleoyl. Additional exemplary lipids, in certainembodiments, include, without limitation, those described in Kim et al.(2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein byreference. Such lipids include, in some embodiments, plant lipids foundto improve liver transfection with mRNA (e.g., DGTS).

Other examples of non-cationic lipids suitable for use in the lipidnanoparticles include, without limitation, nonphosphorous lipids suchas, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate,glycerol ricinoleate, hexadecyl stereate, isopropyl myristate,amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-arylsulfate polyethyloxylated fatty acid amides, dioctadecyl dimethylammonium bromide, ceramide, sphingomyelin, and the like. Othernon-cationic lipids are described in WO2017/099823 or U.S. Pat.Publication US2018/0028664, the contents of which is incorporated hereinby reference in their entirety.

In some embodiments, the non-cationic lipid is oleic acid or a compoundof Formula I, II, or IV of US2018/0028664, incorporated herein byreference in its entirety. The non-cationic lipid can comprise, forexample, 0-30% (mol) of the total lipid present in the lipidnanoparticle. In some embodiments, the non-cationic lipid content is5-20% (mol) or 10-15% (mol) of the total lipid present in the lipidnanoparticle. In embodiments, the molar ratio of ionizable lipid to theneutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1,4:1, 5:1, 6:1, 7:1, or 8:1).

In some embodiments, the lipid nanoparticles do not comprise anyphospholipids.

In some aspects, the lipid nanoparticle can further comprise acomponent, such as a sterol, to provide membrane integrity. Oneexemplary sterol that can be used in the lipid nanoparticle ischolesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5a-cholestanol,53-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, andcholesteryl decanoate; and mixtures thereof. In some embodiments, thecholesterol derivative is a polar analogue, e.g.,cholesteryl-(4′-hydroxy)-buty1 ether. Exemplary cholesterol derivativesare described in PCT publication W02009/127060 and U.S. Pat. PublicationUS2010/0130588, each of which is incorporated herein by reference in itsentirety.

In some embodiments, the component providing membrane integrity, such asa sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%,or 40-50%) of the total lipid present in the lipid nanoparticle. In someembodiments, such a component is 20-50% (mol) 30-40% (mol) of the totallipid content of the lipid nanoparticle.

In some embodiments, the lipid nanoparticle can comprise a polyethyleneglycol (PEG) or a conjugated lipid molecule. Generally, these are usedto inhibit aggregation of lipid nanoparticles and/or provide stericstabilization. Exemplary conjugated lipids include, but are not limitedto, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates,polyamide-lipid conjugates (such as ATTA-lipid conjugates),cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In someembodiments, the conjugated lipid molecule is a PEG-lipid conjugate, forexample, a (methoxy polyethylene glycol)-conjugated lipid.

Exemplary PEG-lipid conjugates include, but are not limited to,PEG-diacylglycerol (DAG) (such asl-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)),PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), apegylated phosphatidylethanoloamine (PEG-PE), PEG succinatediacylglycerol (PEGS-DAG) (such as4-0-(2′,3′-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam,N-(carbonyl-methoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or amixture thereof. Additional exemplary PEG-lipid conjugates aredescribed, for example, in US5,885,613, US6,287,591, US2003/0077829,US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125,US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, thecontents of all of which are incorporated herein by reference in theirentirety. In some embodiments, a PEG-lipid is a compound of Formula III,III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018/0028664, the contentof which is incorporated herein by reference in its entirety. In someembodiments, a PEG-lipid is of Formula II of US20150376115 orUS2016/0376224, the content of both of which is incorporated herein byreference in its entirety. In some embodiments, the PEG-DAA conjugatecan be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl,PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can beone or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol,PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide,PEG- dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol(l-[8′-(Cholest-5-en-3[beta]- oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG- DMB(3,4-Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether),and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine—N—[methoxy(polyethyleneglycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine—N—[methoxy(polyethyleneglycol)-2000]. In some embodiments, the PEG-lipid comprises a structureselected from:

In some embodiments, lipids conjugated with a molecule other than a PEGcan also be used in place of PEG-lipid. For example, polyoxazoline(POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipidconjugates), and cationic-polymer lipid (GPL) conjugates can be used inplace of or in addition to the PEG-lipid.

Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates,ATTA-lipid conjugates and cationic polymer-lipids are described in thePCT and LIS patent applications listed in Table 2 of WO2019051289A9, thecontents of all of which are incorporated herein by reference in theirentirety.

In some embodiments, the PEG or the conjugated lipid can comprise 0-20%(mol) of the total lipid present in the lipid nanoparticle. In someembodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5%(mol) of the total lipid present in the lipid nanoparticle. Molar ratiosof the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugatedlipid can be varied as needed. For example, the lipid particle cancomprise 30-70% ionizable lipid by mole or by total weight of thecomposition, 0-60% cholesterol by mole or by total weight of thecomposition, 0-30% non-cationic-lipid by mole or by total weight of thecomposition and 1-10% conjugated lipid by mole or by total weight of thecomposition. Preferably, the composition comprises 30-40% ionizablelipid by mole or by total weight of the composition, 40-50% cholesterolby mole or by total weight of the composition, and 10- 20%non-cationic-lipid by mole or by total weight of the composition. Insome other embodiments, the composition is 50-75% ionizable lipid bymole or by total weight of the composition, 20-40% cholesterol by moleor by total weight of the composition, and 5 to 10% non-cationic-lipid,by mole or by total weight of the composition and 1-10% conjugated lipidby mole or by total weight of the composition. The composition maycontain 60-70% ionizable lipid by mole or by total weight of thecomposition, 25-35% cholesterol by mole or by total weight of thecomposition, and 5-10% non-cationic-lipid by mole or by total weight ofthe composition. The composition may also contain up to 90% ionizablelipid by mole or by total weight of the composition and 2 to 15%non-cationic lipid by mole or by total weight of the composition. Theformulation may also be a lipid nanoparticle formulation, for examplecomprising 8-30% ionizable lipid by mole or by total weight of thecomposition, 5-30% non-cationic lipid by mole or by total weight of thecomposition, and 0-20% cholesterol by mole or by total weight of thecomposition; 4-25% ionizable lipid by mole or by total weight of thecomposition, 4-25% non-cationic lipid by mole or by total weight of thecomposition, 2 to 25% cholesterol by mole or by total weight of thecomposition, 10 to 35% conjugate lipid by mole or by total weight of thecomposition, and 5% cholesterol by mole or by total weight of thecomposition; or 2-30% ionizable lipid by mole or by total weight of thecomposition, 2-30% non-cationic lipid by mole or by total weight of thecomposition, 1 to 15% cholesterol by mole or by total weight of thecomposition, 2 to 35% conjugate lipid by mole or by total weight of thecomposition, and 1-20% cholesterol by mole or by total weight of thecomposition; or even up to 90% ionizable lipid by mole or by totalweight of the composition and 2-10% non-cationic lipids by mole or bytotal weight of the composition, or even 100% cationic lipid by mole orby total weight of the composition. In some embodiments, the lipidparticle formulation comprises ionizable lipid, phospholipid,cholesterol and a PEG-ylated lipid in a molar ratio of 50: 10:38.5: 1.5.In some other embodiments, the lipid particle formulation comprisesionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of60:38.5: 1.5.

In some embodiments, the lipid particle comprises ionizable lipid,non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol)and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20to 70 mole percent for the ionizable lipid, with a target of 40-60, themole percent of non-cationic lipid ranges from 0 to 30, with a target of0 to 15, the mole percent of sterol ranges from 20 to 70, with a targetof 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to6, with a target of 2 to 5.

In some embodiments, the lipid particle comprises ionizable lipid /non-cationic- lipid / sterol / conjugated lipid at a molar ratio of50:10:38.5: 1.5.

In an aspect, the disclosure provides a lipid nanoparticle formulationcomprising phospholipids, lecithin, phosphatidylcholine andphosphatidylethanolamine.

In some embodiments, one or more additional compounds can also beincluded. Those compounds can be administered separately, or theadditional compounds can be included in the lipid nanoparticles of theinvention. In other words, the lipid nanoparticles can contain othercompounds in addition to the nucleic acid or at least a second nucleicacid, different than the first. Without limitations, other additionalcompounds can be selected from the group consisting of small or largeorganic or inorganic molecules, monosaccharides, disaccharides,trisaccharides, oligosaccharides, polysaccharides, peptides, proteins,peptide analogs and derivatives thereof, peptidomimetics, nucleic acids,nucleic acid analogs and derivatives, an extract made from biologicalmaterials, or any combinations thereof.

In some embodiments, the LNPs comprise biodegradable, ionizable lipids.In some embodiments, the LNPs comprise(9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g.,lipids of WO2019/067992, WO/2017/173054, WO2015/095340, andWO2014/136086, as well as references provided therein. In someembodiments, the term cationic and ionizable in the context of LNPlipids is interchangeable, e.g., wherein ionizable lipids are cationicdepending on the pH.

In some embodiments, the average LNP diameter of the LNP formulation maybe between 10s of nm and 100 s of nm, e.g., measured by dynamic lightscattering (DLS). In some embodiments, the average LNP diameter of theLNP formulation may be from about 40 nm to about 150 nm, such as about40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNPdiameter of the LNP formulation may be from about 50 nm to about 100 nm,from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, fromabout 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nmto about 80 nm, from about 60 nm to about 70 nm, from about 70 nm toabout 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90nm, or from about 90 nm to about 100 nm. In some embodiments, theaverage LNP diameter of the LNP formulation may be from about 70 nm toabout 100 nm. In a particular embodiment, the average LNP diameter ofthe LNP formulation may be about 80 nm. In some embodiments, the averageLNP diameter of the LNP formulation may be about 100 nm. In someembodiments, the average LNP diameter of the LNP formulation ranges fromabout I mm to about 500 mm, from about 5 mm to about 200 mm, from about10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mmto about 60 mm, from about 30 mm to about 55 mm, from about 35 mm toabout 50 mm, or from about 38 mm to about 42 mm.

A LNP may, in some instances, be relatively homogenous. A polydispersityindex may be used to indicate the homogeneity of a LNP, e.g., theparticle size distribution of the lipid nanoparticles. A small (e.g.,less than 0.3) polydispersity index generally indicates a narrowparticle size distribution. A LNP may have a polydispersity index fromabout 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19,0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, thepolydispersity index of a LNP may be from about 0.10 to about 0.20.

The zeta potential of a LNP may be used to indicate the electrokineticpotential of the composition. In some embodiments, the zeta potentialmay describe the surface charge of an LNP. Lipid nanoparticles withrelatively low charges, positive or negative, are generally desirable,as more highly charged species may interact undesirably with cells,tissues, and other elements in the body. In some embodiments, the zetapotential of a LNP may be from about -10 mV to about +20 mV, from about-10 mV to about +15 mV, from about -10 mV to about +10 mV, from about-10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV toabout +15 mV, from about -5 mV to about +10 mV, from about -5 mV toabout +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about+20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV,from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

The efficiency of encapsulation of a protein and/or nucleic acid,describes the amount of protein and/or nucleic acid that is encapsulatedor otherwise associated with a LNP after preparation, relative to theinitial amount provided. The encapsulation efficiency is desirably high(e.g., close to 100%). The encapsulation efficiency may be measured, forexample, by comparing the amount of protein or nucleic acid in asolution containing the lipid nanoparticle before and after breaking upthe lipid nanoparticle with one or more organic solvents or detergents.An anion exchange resin may be used to measure the amount of freeprotein or nucleic acid (e.g., RNA) in a solution. Fluorescence may beused to measure the amount of free protein and/or nucleic acid (e.g.,RNA) in a solution. For the lipid nanoparticles described herein, theencapsulation efficiency of a protein and/or nucleic acid may be atleast 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments,the encapsulation efficiency may be at least 80%. In some embodiments,the encapsulation efficiency may be at least 90%. In some embodiments,the encapsulation efficiency may be at least 95%.

A LNP may optionally comprise one or more coatings. In some embodiments,a LNP may be formulated in a capsule, film, or table having a coating. Acapsule, film, or tablet including a composition described herein mayhave any useful size, tensile strength, hardness or density.

Additional exemplary lipids, formulations, methods, and characterizationof LNPs are taught by WO2020/061457 and WO2021/113777, each of which isincorporated herein by reference in its entirety. Further exemplarylipids, formulations, methods, and characterization of LNPs are taughtby Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater(2021). doi.org/10.1038/s41578-021-00358-0, which is incorporated hereinby reference in its entirety (see, for example, exemplary lipids andlipid derivatives of FIG. 2 of Hou et al.).

In some embodiments, in vitro or ex vivo cell lipofections are performedusing Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNATransfection Reagent (Mirus Bio). In certain embodiments, LNPs areformulated using the GenVoy_ILM ionizable lipid mix (PrecisionNanoSystems). In certain embodiments, LNPs are formulated using2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) ordilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), theformulation and in vivo use of which are taught in Jayaraman et al.Angew Chem Int Ed Engl 51 (34):8529-8533 (2012), incorporated herein byreference in its entirety.

LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g.,Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 andWO2019067910, both incorporated by reference, and are useful fordelivery of circular polyribonucleotides and linear polyribonucleotidesdescribed herein.

Additional specific LNP formulations useful for delivery of nucleicacids (e.g., circular polyribonucleotides, linear polyribonucleotides)are described in US8158601 and US8168775, both incorporated byreference, which include formulations used in patisiran, sold under thename ONPATTRO.

Exemplary dosing of polyribonucleotide (e.g., a circularpolyribonucleotide, a linear polyribonucleotide) LNP may include about0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA).Exemplary dosing of AAV comprising a polyribonucleotide (e.g., acircular polyribonucleotide, a linear polyribonucleotide) may include anMOI of about 10¹¹, 10¹², 10¹³, and 10¹⁴ vg/kg.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the disclosure and are not intended to limit thescope of what the inventors regard as their invention.

Example 1: Design of Anabaena Self-Splicing Permuted Intron-Exon (PIE)Construct With Extended Annealing Region

This example describes the design of Anabaena self-splicing permutedintron-exon (PIE) sequences with extended annealing region to providebetter circularization efficiency.

Schematics depicting exemplary designs of DNA constructs are provided inFIG. 1A and FIG. 1B. In this example, the constructs include, from 5′ to3′: a 3′ half of group I catalytic intron fragment (Anabaena 3′half-intron), a 3′ splice site, a 3′ exon fragment (Anabaena E2), aspacer element, a polynucleotide cargo, a 5′ exon fragment (AnabaenaE1), a 5′ splice site, and a 5′ half of group I catalytic intronfragment (Anabaena 5′ half-intron). E2 has a 5 nucleotide complementarysequence (5′-TCCGT-3′) (SEQ ID NO: 1) to E1 (5′-ACGGA-3′) (SEQ ID NO: 2)(FIGS. 1A and 1B, black lines on the E2 and E1). To generate a constructthat has an extended annealing region between E2 and E1, 5 nucleotidesfrom E2 were mutated to have an extended 7 nucleotide annealing regionwith E1 (TGACCTT (SEQ ID NO: 3) ➔ AGCGTCT (SEQ ID NO: 4), bold characterrepresents mutated sequences) (FIG. 1B, gray line on the E2 and E1,asterisks in E2 represent mutation on the sequence). The total annealingregion from Anabaena permuted intron-exon (PIE) with an extendedannealing region is 12 nucleotides (E2; 5′-TCCGTAGCGTCT-3′ (SEQ ID NO:5), E1; 5′-AGACGCTACGGA-3′ (SEQ ID NO: 6)) (FIG. 1B).

The RNA structure was estimated by RNA structure prediction tool, RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). Extensionof E2-E1 interaction was generated by modifying sequence results inproper E2-E1 interaction and condensed self-splicing intron structure(FIGS. 2A and 2B).

Constructs that have the Anabaena PIE with an annealing region of 5nucleotides (Anabaena 1) and annealing sequences with an extendedannealing region (Anabaena 2) were designed to compare circularizationefficiency. Anabaena PIE constructs described in Wesselhoeft, et al.2018 (Nat. Commun. 9:2629) (Anabaena 3) were also used for comparison.In this example, the constructs were designed to include polyA50 as thespacer element, and a combination of an EMCV internal ribosome entrysite (IRES) and an ORF as the polynucleotide cargo. Two different ORFswere tested: a Gaussia luciferase (Gluc) ORF (558 nucleotides) and aSARS-CoV-2 spike protein ORF (3822nts). The size of circular RNA was 1.2Kb with the Gluc ORF and 4.5 Kb with the SARS-CoV-2 spike protein ORF.

Unmodified linear RNA was synthesized by in vitro transcription using T7RNA polymerase from a DNA template in the presence of 7.5 mM of NTP.Template DNA was removed by treating with DNase for 20 minutes.Synthesized linear RNA was purified with an RNA clean up kit (NewEngland Biolabs, T2050). Self-splicing occurred during transcription; noadditional reaction was required. To monitor self-splicing efficiency,200 ng of column purified in vitro transcribed RNA was mixed with gelloading buffer II (Thermo Fisher, AM8546G) and heated at 95° C. for 3minutes, then incubated on ice for 3 minutes. The samples were thenseparated by 6% Urea polyacrylamide gel electrophoresis (Urea PAGE), andthe RNA band was stained using gel stain and visualized using an imagingsystem. Extending the annealing sequence from 5 nucleotides to 12nucleotides increased circularization efficiency up to two-fold andshowed similar circularization efficiency with Anabaena 3 in the case ofthe 1.2 Kb circular RNA (FIG. 3A). In the case of the 4.5 Kb circularRNA, Anabaena PIE with an extended annealing region (Anabaena 2) showed40% better circularization efficiency as compared to Anabaena 3 andthree-fold higher than Anabaena PIE with an annealing region of 5nucleotides (Anabaena 1) (FIG. 3B).

Anabaena PIE designed to have an extended E2-E1 annealing sequence(Anabaena 2) showed 2-3-fold better circularization efficiency thanAnabaena PIE with an annealing region of 5 nucleotides regardless of thesize of the circular RNA (Anabaena 1). This shows similarcircularization efficiency for the easier-to circularize 1.2 Kbconstruct and 40% better circularization efficiency for the difficult4.5 Kb construct.

Example 2: Protein Expression From Circular RNA Generated by AnabaenaSelf-Splicing PIE With an Extended Annealing Region

This example demonstrates protein expression from circular RNA generatedby Anabaena self-splicing PIE with an extended annealing region.

In this example, constructs having Anabaena PIE with an annealing regionof 5 nucleotides (Anabaena 1) and extended annealing sequences (Anabaena2) were designed as described in Example 1 to compare proteinexpression. In this example, the constructs were designed to includepolyA50 as the spacer element, and a combination of an EMCV IRES and anORF as the polynucleotide cargo. Two different ORFs were tested: Gluc(558nts) and SARS-CoV-2 spike protein (3822nts). Anabaena 3, asdescribed in Example 1, was also tested for comparison.

Linear RNA was synthesized by in vitro transcription using T7 RNApolymerase in the presence of 7.5 mM of NTP. Template DNA was removed bytreating with DNase for 20 minutes. Synthesized linear RNA was purifiedwith an RNA clean up kit (New England Biolabs, T2050). Circular RNAencoding Gluc was purified by Urea PAGE, eluted in a buffer (0.5 MSodium Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated andresuspended in RNAse-free water. Circular RNA encoding spike proteinfrom SARS-CoV-2 was purified by reverse phase chromatography and thefractions were buffer exchanged with sodium citrate and then waterthrough ultrafiltration using Amicon Ultra Centrifugal filters (SigmaAldrich).

To compare expression of circular RNA encoding Gluc, circular RNAgenerated by Anabaena 1 and Anabaena 2 were prepared. For comparison,circular RNA produced by Anabaena 3 was also prepared. HeLa cells(10,000 cells per well in a 96 well plate) were transfected with 0.1pmole of purified circular RNAs using LIPOFECTAMINE® MessengerMAXtransfection reagent (Invitrogen) according to the manufacturer’sprotocol. Cell culture media was harvested and replaced with fresh mediaat 24 hr, 48 hr and 72 hr timepoints to measure Gluc activity. Tomeasure Gluc activity, 10 µl of harvested cell media was transferred toa white 96 well plate, and a bioluminescent reporter assay system wasused according to the manufacturer’s instruction (Pierce GaussiaLuciferase Flash Assay Kit, 16158, Thermo Scientific). The plate wasread in a luminometer instrument (Promega).

To compare expression of circular RNA encoding SARS-CoV-2 spike protein,circular RNA generated by Anabaena PIE with an annealing region of 5nucleotides (Anabaena 1) and Anabaena PIE with an extended annealingregion (Anabaena 2) were prepared. For comparison, circular RNA producedby Anabaena 3 was also prepared. HeLa cells (1.2 million cells per wellin a 6 well plate) were transfected with 4 pmol of purified circular RNAusing LIPOFECTAMINE® MessengerMAX (Invitrogen) transfection agentaccording to manufacturer’s instructions. After 48 hour transfection,cells were harvested by trypsinization and resuspended in coldserum-free media. Cells were then stained with anti-SARS-CoV-2 RBDantibody for one hour and subsequently incubated with anti-mouse IgG1antibody AF647 for 30 minutes. The stained population was measured byflow cytometry.

Circular RNA generated by Anabaena PIE with an extended annealing region(Anabaena 2) showed similar expression with circular RNA generated byAnabaena PIE with an annealing region of 5 nucleotides (Anabaena 1) andAnabaena 3-produced circular RNA when encoding Gluc as a polynucleotidecargo (FIG. 4 ). In the case of circular RNA encoding SARS-CoV-2 spikeprotein, circular RNA generated by Anabaena 2 showed around three-foldbetter expression than Anabaena 3-produced circular RNA and 50% moreexpression than Anabaena 1 generated circular RNA (FIG. 5 ).

Example 3: The Effect of the Length of Annealing Region onCircularization Efficiency in Anabaena Self-Splicing PIE

This example demonstrates the effect of the length of annealing regionon the circularization efficiency in Anabaena self-splicing PIE.

In Example 1 above, we showed that extending the annealing region from 5nucleotides to 12 nucleotides by mutating the E2 sequence augmentscircularization efficiency of Anabaena PIE. To examine the effect of thelength of the annealing region on circularization efficiency, threeadditional constructs were designed to have a further extended annealingregion between E2 and E1 by including additional sequences at the 5′ endof E1 that are complementary to E2: (1) 5 nucleotide extension(5′-CGTTT-3′) (SEQ ID NO: 7), (2) 10 nucleotide extension (5′-ACGACCGTTT-3′) (SEQ ID NO: 8), and (3) 15 nucleotide extension (5′-CCCACACGACCGTTT-3′) (SEQ ID NO: 9). The complementary sequence in E2 isa 5 nucleotide extension (5′- AAACG-3′) (SEQ ID NO: 10), 10 nucleotideextension (5′-AAACGGTCGT-3′) (SEQ ID NO: 11), or 15 nucleotide extension(5′- AAACGGTCGTGTGGG-3′) (SEQ ID NO: 12), respectively. Total annealingsequence is 17 nucleotides, 22 nucleotides, or 27 nucleotides,respectively. A schematic depicting exemplary designs of DNA constructswith extended annealing regions between E2 and E1 is provided in FIG. 6. Constructs with extended annealing sequences (Anabaena 2) and extendedannealing region (5 nucleotide extension, 10 nucleotide extension and 15nucleotide extension) were designed to compare circularizationefficiency. In this example, the constructs were designed to includepolyA50 as the spacer element, and a combination of an EMCV IRES andGluc as the polynucleotide cargo. Linear RNA was synthesized by in vitrotranscription using T7 RNA polymerase in the presence of 7.5 mM of NTP.Template DNA was removed by treating with DNase for 20 minutes.Synthesized linear RNA was purified with an RNA clean up kit (NewEngland Biolabs, T2050).

Self-splicing occurred during transcription; no additional reaction wasrequired. To monitor self-splicing efficiency, 200 ng of column purifiedin vitro transcribed RNA was mixed with gel loading buffer II (ThermoFisher, AM8546G) and heated at 95° C. for 3 minutes, then incubated onice for 3 minutes. The samples were then separated by 6% Urea PAGE, andthe RNA band was stained using gel stain and visualized using an imagingsystem.

Further extending of the annealing region between E2 and E1 (5 ntsextension, 10 nts extension, or 15 nts extension) showed comparablecircularization efficiency with Anabaena PIE with an extended annealingregion (Anabaena 2) (FIG. 7 ). A 15 nucleotide extension of annealingregion showed 30% better circularization efficiency compared withAnabaena 2 (FIG. 7 ). This data indicates that the E2-E1 interaction isimportant for efficient circularization and further extending theannealing region can increase circularization efficiency.

Example 4: Protein Expression From Circular RNA Generated by AnabaenaSelf-Splicing PIE With Extended Annealing Sequence

This example demonstrates protein expression from circular RNA generatedby Anabaena self-splicing PIE with extended annealing sequence.

In this example, constructs with extended annealing sequences (Anabaena2) and extended annealing region (5 nucleotide extension, 10 nucleotideextension, and 15 nucleotide extension) were designed as described inExample 3 to compare protein expression. In this example, the constructswere designed to include polyA50 as the spacer element, and acombination of an EMCV IRES and Gluc as the polynucleotide cargo.

Linear RNA was synthesized by in vitro transcription using T7 RNApolymerase in the presence of 7.5 mM of NTP. Template DNA was removed bytreating with DNase for 20 minutes. Synthesized linear RNA was purifiedwith an RNA clean up kit (New England Biolabs, T2050). Circular RNAencoding Gluc was purified by Urea PAGE, eluted in a buffer (0.5 MSodium Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated, andresuspended in RNAse-free water.

To compare expression of circular RNA encoding Gluc, circular RNAgenerated by Anabaena 2 and Anabaena PIE with a further extendedannealing region (5 nucleotide extension, 10 nucleotide extension, or 15nucleotide extension) were prepared as described in Example 3. HeLacells (10,000 cells per well in a 96 well plate) were transfected with0.1 pmole of purified circular RNAs using LIPOFECTAMINE® MessengerMAX(Invitrogen) transfection agent according to manufacturer’sinstructions. Transfectants were prepared for each time pointsseparately. At 6 hours, 24 hours and 48 hours, culture media washarvested. To measure Gluc activity, 10 µl of harvested cell media wastransferred to a white 96 well plate and a bioluminescent reporter assaysystem was used according to the manufacturer’s instruction (PierceGaussia Luciferase Flash Assay Kit, 16158, Thermo Scientific). The platewas read in a luminometer instrument (Promega).

Circular RNA generated by Anabaena PIE with a further extended annealingregion (5 nts extension, 10 nts extension, or 15 nts extension) showedsimilar or better expression than that of circular RNA generated byAnabaena 2 (FIG. 8 ). For example, circular RNA generated by AnabaenaPIE with a 15 nucleotide extension (27 nucleotides total) showedthree-fold higher expression than Anabaena PIE with an extendedannealing region of 12 nucleotides (Anabaena 2). This data indicatesthat annealing region extension is important for not onlycircularization efficiency but also for expression.

Example 5: Design of Tetrahymena Self-Splicing Permuted Intron-Exon(PIE) With Extended Annealing Region

This example describes the design of Tetrahymena self-splicing permutedintron-exon (PIE) with extended annealing region.

Schematics depicting exemplary designs of DNA constructs are provided inFIG. 9A and FIG. 9B.

In this example, the constructs include, from 5′-to-3′: a 3′ half ofgroup I catalytic intron fragment (Tetrahymena 3′ half-intron), a 3′splice site, a 3′ exon fragment (Tetrahymena E2), a spacer element, apolynucleotide cargo, a 5′ exon fragment (Tetrahymena E1), a 5′ splicesite, and a 5′ half of group I catalytic intron fragment (Tetrahymena 5′half-intron). E2 has a 6 nucleotide complementary sequence (5′-AAGGTA-3′) (SEQ ID NO: 13) to the 5′ half-intron (5′- TACCTT-3′) (SEQ IDNO: 14) that forms helix P10 (FIG. 9 , black lines on E1 and 5′half-intron). To generate a construct that has an extended annealingregion between E2 and 5′ half-intron, 6 nucleotides were added to the 3′end of the annealing region in E2 (5′- AATATT-3′ (SEQ ID NO: 15), graybox on E2 in FIGS. 9A and 9B). The total annealing region fromTetrahymena self-splicing PIE with extended annealing region is 12nucleotides (E2; 5′-AAGGTAAATATT-3′ (SEQ ID NO: 16), 5′intron; 5′-AATATTTACCTT-3′ (SEQ ID NO: 17), bold characters represent extendedannealing region) (FIG. 9B).

The RNA structure was estimated by the RNA structure prediction tool,RNA fold (rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi).Extension of E2-5′ half-intron interaction by additional sequenceresulted in proper helix P10 formation and condensed self-splicingintron structure (FIGS. 10A and 10B).

Constructs that have a Tetrahymena permuted intron-exon with anannealing region of 6 nucleotides (Tetrahymena 1) and extended annealingsequences (Tetrahymena 2) were designed to compare circularizationefficiency. In this example, the constructs were designed to includepolyA50 as the spacer element, and a combination of an EMCV IRES andhEPO ORF as the polynucleotide cargo. The size of the circular RNA was1.2 Kb.

Linear RNA was synthesized by in vitro transcription using T7 RNApolymerase in the presence of 7.5 mM of NTP. Template DNA was removed bytreating with DNase for 20 minutes. Synthesized linear RNA was purifiedwith an RNA clean up kit (New England Biolabs, T2050). Self-splicingoccurred during transcription; no additional reaction was required. Tomonitor circularization efficiency, 200 ng of column purified in vitrotranscribed RNA was mixed with gel loading buffer II (Thermo Fisher,AM8546G) and heated at 95° C. for 3 minutes, then incubated on ice for 3minutes. The samples were then separated by 6% Urea PAGE, and the RNAband was stained using gel stain and visualized using an imaging system.Extending the annealing sequence from 6 nucleotides to 12 nucleotides(Tetrahymena 2) showed similar circularization efficiency with aTetrahymena self-splicing PIE with an annealing region of 6 nucleotides(Tetrahymena 1) (FIG. 11 ). This data indicate that circularization wasnot disrupted by extension of the annealing sequence in Tetrahymenaself-splicing PIE.

Example 6: Protein Expression From Circular RNA Generated by TetrahymenaPIE With an Extended Annealing Region

This example describes protein expression from circular RNA generated byTetrahymena self-splicing PIE with an extended annealing region.

To compare protein expression, DNA constructs having a Tetrahymenaself-splicing PIE with an annealing region of 6 nucleotides(Tetrahymena 1) and extended annealing sequences (Tetrahymena 2) aredesigned as described in Example 5. The constructs are designed toinclude a polyA50 as the spacer element, and a combination of an EMCVIRES, and Gluc ORF as the polynucleotide cargo.

Linear RNA is synthesized by in vitro transcription using T7 RNApolymerase in the presence of 7.5 mM of NTP. Template DNA is removed bytreating with DNase. Synthesized linear RNA is purified with an RNAclean up kit (New England Biolabs, T2050). Circular RNA encoding Gluc ispurified by Urea PAGE, eluted in a buffer (0.5 M Sodium Acetate, 0.1%SDS, 1 mM EDTA), ethanol precipitated, and resuspended in RNAse-freewater.

To compare expression of circular RNA encoding Gluc, circular RNAgenerated by Tetrahymena PIE with an annealing region of 6 nucleotides(Tetrahymena 1) and Tetrahymena PIE with an extended annealing region(Tetrahymena 2) are prepared as described above. HeLa cells (10,000cells per well in a 96 well plate) are transfected with 0.1 pmoles ofpurified circular RNA using LIPOFECTAMINE® MessengerMAX (Invitrogen)transfection agent according to manufacturer’s instructions.Transfectants are prepared for each time points separately. At 6 hours,24 hours and 48 hours, culture media is harvested. To measure Glucactivity, harvested cell media is transferred to a white 96 well plateand a bioluminescent reporter assay system is used according tomanufacturer’s instructions. The plate is read in a luminometerinstrument.

Example 7: Design of T4 Phage Self-Splicing Permuted Intron-Exon (PIE)With Extended Annealing Region

This example describes the design of T4 phage self-splicing PIE withextended annealing region.

Schematics depicting exemplary designs of DNA constructs are provided inFIG. 12A and FIG. 12B. The construct includes, from 5′-to-3′: a 3′ halfof group I catalytic intron fragment (T4 phage 3′ half-intron), a 3′splice site, a 3′ exon fragment (T4 phage E2), a spacer element, apolynucleotide cargo, a 5′ exon fragment (T4 phage E1), a 5′ splicesite, and a 5′ half of group I catalytic intron fragment (T4 phage 5′half-intron). E2 has a 2 nucleotide complementary sequence (5′- CT-3′)to the 5′ half-intron (5′- AG-3′) that forms helix P10 (FIGS. 12A and12B, black lines on E2 and 5′ half-intron). To generate a construct thathas an extended annealing region between E2 and 5′ half-intron, 4nucleotides from E2 were mutated to have an extended 5 nucleotideannealing region with the 5′ half-intron (5′-ACCGT-3′ (SEQ ID NO: 18) ➔5′-CAATT-3′ (SEQ ID NO: 19), bold characters represent mutatedsequences). The total annealing region from T4 phage PIE with anextended annealing region is 7 nucleotides (E2; 5′-CTCAATT-3′ (SEQ IDNO: 20), 5′ half-intron; 5′-AATTGAG -3′ (SEQ ID NO: 21), bold charactersrepresent extended annealing sequences) (FIGS. 12A and 12B). To comparecircularization efficiency, constructs that have T4 phage PIE with anannealing region of 2 nucleotides (T4 phage 1) and extended annealingsequences (T4 phage 2) were designed. In this example, the constructswere designed to include polyA50 as the spacer element, a combination ofan EMCV IRES and Gluc ORF as the polynucleotide cargo. The size of thecircular RNA was 1.2 K. Linear RNA was synthesized by in vitrotranscription using T7 RNA polymerase in the presence of 7.5 mM of NTP.Template DNA was removed by treating with DNase. Synthesized linear RNAwas purified with an RNA clean up kit (New England Biolabs, T2050).

Self-splicing occurred during transcription; no additional reaction isrequired. To monitor circularization efficiency, 200 ng of columnpurified in vitro transcribed RNA was mixed with gel loading buffer II(Thermo Fisher, AM8546G) and heated at 95° C. for 3 minutes, thenincubated on ice for 3 minutes. The samples were then separated by 6%Urea PAGE, and the RNA band was stained using gel stain and visualizedusing an imaging system.

Extending the annealing sequence (T4 phage 2) showed similarcircularization efficiency with a T4 phage self-splicing PIE with anannealing region of 6 nucleotides (T4 phage 1) (FIG. 13 ). This dataindicate that circularization was not disrupted by extension of theannealing sequence in T4 phage self-splicing PIE.

Example 8: Protein Expression From Circular RNA Generated by T4 PhageSelf-Splicing PIE With an Extended Annealing Region

This example describes expression of circular RNA generated by T4 phageself-splicing PIE with an extended annealing region.

To compare protein expression, DNA constructs with T4 phage PIE with anannealing region of 2 nucleotides (T4 phage 1) and extended annealingsequences (T4 phage 2) are designed as described in Example 7. In thisexample, the constructs are designed to include polyA50 as the spacerelement, and a combination of an EMCV IRES and Gluc ORF as thepolynucleotide cargo.

Linear RNA is synthesized by in vitro transcription using T7 RNApolymerase in the presence of 7.5 mM of NTP. Template DNA is removed bytreating with DNase. Synthesized linear RNA is purified with an RNAclean up kit (New England Biolabs, T2050). Circular RNA encoding Gluc ispurified by Urea PAGE, eluted in a buffer (0.5 M Sodium Acetate, 0.1%SDS, 1 mM EDTA), ethanol precipitated, and resuspended in RNAse-freewater.

To compare expression of circular RNA encoding Gluc, circular RNAgenerated by T4 phage PIE with an annealing region of 2 nucleotides (T4phage 1) and T4 phage PIE with an extended annealing region (T4 page 2)are prepared as described above. HeLa cells (10,000 cells per well in a96 well plate) are transfected with 0.1 pmole of purified circular RNAsusing LIPOFECTAMINE® MessengerMAX (Invitrogen) transfection agentaccording to manufacturer’s instructions. Transfectants are prepared foreach time point separately. At 6 hours, 24 hours and 48 hours, culturemedia is harvested. To measure Gluc activity, harvested cell media istransferred to a white 96 well plate and a bioluminescent reporter assaysystem is used according to the manufacturer’s instructions. The plateis read in a luminometer instrument.

Example 9: Design of Self-Splicing Permuted Intron-Exon (PIE) ConstructWith Extended Annealing Region

This example describes the design of various self-splicing permutedintron-exon (PIE) sequences with extended annealing region to providebetter circularization efficiency.

Schematics depicting exemplary designs of DNA constructs are provided inFIG. 14A and FIG. 14B. In this example, the constructs include, from 5′to 3′: a 3′ half of group I catalytic intron fragment (3′ half-intron),a 3′ splice site, a 3′ exon fragment (E2), a spacer element, apolynucleotide cargo, a 5′ exon fragment (E1), a 5′ splice site, and a5′ half of group I catalytic intron fragment (5′ half-intron).

Different group I introns have different lengths of complementarysequence (FIG. 19 ). For example, E2 of Synechococcus elongatus PCC 6301has a 7 nucleotide complementary sequence to E1 of Synechococcuselongatus PCC 6301; E2 of Anabaena azollae, Anabaena cylindrica, andScytonema hofmanni have 5 nucleotides of complementary sequences to E1of Anabaena azollae, Anabaena cylindrica, and Scytonema hofmanni,respectively. To generate a construct that has an extended annealingregion between E2 and E1, sequences in E2 were mutated to have anextended annealing region with E1 as described in FIG. 19 . The totalannealing region from group I permuted intron-exon (PIE) with anextended annealing region is 17 nucleotides.

Original (1) and extended (2) annealing regions from FIG. 19 are asfollows:

-   Synechococcus 1 TCCGCTGACTGTAAAGG (SEQ ID NO: 92)-   Synechococcus 2 TCCGCTGCGTCTACCGT (SEQ ID NO: 93)-   Anabaena azollae 1 TCCGTTGACTGTAAAAA (SEQ ID NO: 94)-   Anabaena azollae 2 TCCGTAGCGTCTACCAT (SEQ ID NO: 95)-   Anabaena cylindrica 1 TCCGTTGACCTTAAACG (SEQ ID NO: 96)-   Anabaena cylindrica 2 TCCGTAGCGTCTACCAT (SEQ ID NO: 97)-   Scytonema 1 CCCGAAGGTCAGTGGTT (SEQ ID NO: 98)-   Scytonema 2 CCCGACGAGCTACCAGG (SEQ ID NO: 99)

The RNA structures were estimated by RNA structure prediction tool, RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). Extensionof E2-E1 interaction was generated by modifying sequence results inproper E2-E1 interaction and condensed self-splicing intron structure(FIGS. 15A-15B, 16A-16B, 17A-17B, and 18A-18B).

Constructs that have the PIE with an original annealing region andannealing sequences with an extended annealing region were designed tocompare circularization efficiency. For comparison, Anabaena 1 andAnabaena 2 constructs were also used. In this example, the constructswere designed to include a spacer element, and a combination of an EMCVIRES and a 3822 nucleotide ORF as the polynucleotide cargo. The size ofthe circular RNA was 4.5 Kb.

Unmodified linear RNA was synthesized by in vitro transcription using T7RNA polymerase from a DNA template in the presence of 12.5 mM of NTP.Template DNA was removed by treating with DNase for 20 minutes.Synthesized linear RNA was purified with an RNA clean up kit (NewEngland Biolabs, T2050). Self-splicing occurred during transcription; noadditional reaction was required. To monitor self-splicing efficiency,column purified in vitro transcribed RNA was separated on an anionicexchange (AEX) column through HPLC. The percentage of linear andcircular peaks were measured, and circularization efficiency wasnormalized with that of constructs that have the PIE with the originalannealing region.

Extending the annealing sequence increased circularization efficiency upto five-fold for Anabaena (Anabaena 2), Synechococcus elongatus PCC 6301(Synechococcus 2), and Anabaena cylindrica (Anabaena cyclindrica 2), andup to ten-fold for Anabaena azollae (Anabaena azollae 2), but noincrease in circularization efficiency was observed for Scytonemahofmanni (Scytonema 2) (FIG. 20 ). This shows that circularizationefficiency can be increased by modifying other group I introns using thesame or similar strategy as described herein for Anabaena intron.

Example 10: Design of Anabaena Self-Splicing Permuted Intron-exon (PIE)Construct With Extended Stem Region to Enhance End to End Interaction

This example describes the design of Anabaena self-splicing permutedintron-exon (PIE) sequences with extended stem region to provide bettercircularization efficiency by enhancing end to end interaction.

Schematics depicting exemplary designs of DNA constructs are provided inFIG. 21B. In this example, the constructs include, from 5′ to 3′: a 3′half of group I catalytic intron fragment (Anabaena 3′ half-intron), a3′ splice site, a 3′ exon fragment (Anabaena E2), a spacer element, apolynucleotide cargo, a 5′ exon fragment (Anabaena E1), a 5′ splicesite, and a 5′ half of group I catalytic intron fragment (Anabaena 5′half-intron). Two versions of constructs that have an extended stemregion were designed. For design of Anabaena 4, an additional stemregion (5′-GUAAGUU-3′) was placed next each other. For design ofAnabaena 5, a bulge region in P6b was filled to make a stem.

Unmodified linear RNA was synthesized by in vitro transcription using T7RNA polymerase from a DNA template in the presence of 12.5 mM of NTP.Template DNA was removed by treating with DNase for 20 minutes.Synthesized linear RNA was purified with an RNA clean up kit (NewEngland Biolabs, T2050). Self-splicing occurred during transcription; noadditional reaction was required. To monitor self-splicing efficiency,column purified in vitro transcribed RNA was separated on an anionicexchange (AEX) column through HPLC. The percentage of linear andcircular peaks were measured and circularization efficiency wasnormalized with that of the corresponding original constructs.

Constructs with an extended stem region showed comparablecircularization efficiency with constructs that have the Anabaena PIEwith an extended annealing region (Anabaena 2) and constructs that havethe Anabaena PIE with an annealing region of 5 nucleotides (Anabaena 1)(FIG. 22 ).

Synechococcus elongatus PCC 6301: 3′ half-intron E2

TAAACAACTAACAGCTTTAGAAGGTGCAGAGACTAGACGGGAGCTACCCTAACGGATTCAGCCGAGGGTAAAGGGATAGTCCAATTCTCAACATCGCGATTGTTGATGGCAGCGAAAGTTGCAGAGAGAATGAAAATCCGCTGACTGTAAAGGTCGTGAGGGTTCGAGTCCCTCCGCCCCCA (SEQ ID NO: 80)

Synechococcus elongatus PCC 6301: E1 5′ half-intron

ACGGTAGACGCAGCGGACTTAGAAAACTGGGCCTCGATCGCGAAAGGGATCGAGTGGCAGCTCTCAAACTCAGGGAAACCTAAAACTTTAAACATTMAAGTCATGGCAATCCTGAGCCAAGCTAAAGC (SEQ ID NO: 81)

Anabaena azollae: 3′ half-intron E2

TTAAACTCAAAATTTAAAATCCCAAATTCAAAATTCCGGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTAAAGCCGAGGGTAAAGGGAGAGTCCAATTCTCAAAGCCTGAAGTTGCTGAAGCAACAAGGCAGTAGTGAAAGCTGCGAGAGAATGAAAATCCGTTGACTGTAAAAAGTCGTGGGGGTTCAAGTCCCCCCACCCCC (SEQ ID NO: 82)

Anabaena azollae: E1 5′ half-intron

ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTTAAGTGGAAGCTCTCAAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCAGAAAATTTAGACTTGAGATTTGATTTTGGA G (SEQ ID NO: 83)

Anabaena cylindrica: 3′ half-intron E2

GGCTTTCAATTTGAAATCAGAAATTCAAAATTCAGGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTAAAGGCGAGGGTAAAGGGAGAGTCCAATTCTTAAAGCCTGAAGTTGTGCAAGCAACAAGGCAACAGTGAAAGCTGTGGAAGAATGAAAATCCGTTGACCTTAAACGGTCGTGGGGGTTCAAGTCCCCCCACCCCC (SEQ ID NO: 84)

Anabaena cylindrica: E1 5′ half-intron

ATGGTAGACGCTACGGACTTAGAAAACTGAGCCTTGATAGAGAAATCTTTCAAGTGGAAGCTCTCAAATTCAGGGAAACCTAAATCTGAATACAGATATGGCAATCCTGAGCCAAGCCCGGAAATTTTAGAATCAAGATTTTATTTT (S EQ ID NO: 85)

Scytonema hofmanni: 3′ half-intron E2

AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGGGACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCGAAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA (SEQ ID NO: 86)

Scytonema hofmanni: E1 5′ half-intron

AGAAATGGAGAAGGTGTAGAGACTGGAAGGCAGGCACCCTAACGTTAAAGGCGAGGGTGAAGGGACAGTCCAGACCACAAACCAGTAAATCTGGGCAGCGAAAGCTGTAGATGGTAAGCATAACCCGAAGGTCAGTGGTTCAAATCCACTTCCCGCCACCAAATTAAAAAAACAATAA (SEQ ID NO: 87)

Anabaena 4: 3′ half-intron E2

AACAACAGATAACTTACTAACTTACAGCTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA (SEQ ID N O: 88)

Anabaena 4: E1 5′ half-intron

AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAGTAAGTT (SEQ ID  NO: 89)

Anabaena 5: 3′ half-intron E2

AACAACAGATAACTTACTAGTTACTAGTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGAAAATCCGTAGCGTCTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCA (SEQ ID NO: 9 0)

Anabaena 5: E1 5′ half-intron

AGACGCTACGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT(SEQ ID NO: 91)

OTHER EMBODIMENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.Other embodiments are within the claims.

1. A linear polyribonucleotide having the formula5′—(A)—(B)—(C)—(D)—(E)—(F)—(G)—3′, wherein: (A) comprises a 3′ half ofGroup I catalytic intron fragment; (B) comprises a 3′ splice site; (C)comprises a 3′ exon fragment comprising a first annealing regioncomprising from 8 to 50 ribonucleotides; (D) comprises apolyribonucleotide cargo; (E) comprises a 5′ exon fragment comprising asecond annealing region comprising from 8 to 50 ribonucleotides that canhybridize to the first annealing region; (F) comprises a 5′ splice site;and (G) comprises a 5′ half of Group I catalytic intron fragment.
 2. Thelinear polyribonucleotide of claim 1, wherein the first annealing regioncomprises from 10 to 30 ribonucleotides and the second annealing regioncomprises from 10 to 30 ribonucleotides.
 3. The linearpolyribonucleotide of claim 2 wherein the first annealing regioncomprises 12 ribonucleotides and the second annealing region comprises12 ribonucleotides.
 4. The linear polyribonucleotide of claim 2 whereinthe first annealing region comprises 17 ribonucleotides and the secondannealing region comprises 17 ribonucleotides.
 5. The linearpolyribonucleotide of claim 2 wherein the first annealing regioncomprises 22 ribonucleotides and the second annealing region comprises22 ribonucleotides.
 6. The linear polyribonucleotide of claim 2, whereinthe first annealing region comprises 27 ribonucleotides and the secondannealing region comprises 27 ribonucleotides.
 7. The linearpolyribonucleotide of claim 1, wherein the first annealing region andthe second annealing region comprise zero or one mismatched base pair.8. The linear polyribonucleotide of claim 1, wherein the 3′ half ofGroup I catalytic intron fragment of (A) is the 5′ terminus of thelinear polynucleotide.
 9. The linear polyribonucleotide of claim 1,wherein the 5′ half of Group I catalytic intron fragment of (G) is the3′ terminus of the linear polyribonucleotide.
 10. The linearpolyribonucleotide of claim 1, wherein the linear polyribonucleotidedoes not comprise a further annealing region.
 11. The linearpolyribonucleotide of claim 1, wherein the polyribonucleotide cargo of(D) comprises an expression sequence, a non-coding sequence, or anexpression sequence and a non-coding sequence.
 12. The linearpolyribonucleotide of claim 11, wherein the expression sequence encodesa polypeptide.
 13. The linear polyribonucleotide of claim 12, whereinthe polyribonucleotide cargo of (D) comprises an IRES operably linked tothe expression sequence encoding the polypeptide.
 14. The linearpolyribonucleotide of claim 1, wherein the linear polyribonucleotidefurther comprises a first spacer region between the 3′ exon fragment of(C) and the polyribonucleotide cargo of (D).
 15. The linearpolyribonucleotide of claim 1, wherein the linear polyribonucleotidefurther comprises a second spacer region between the polyribonucleotidecargo of (D) and the 5′ exon fragment of (E).
 16. The linearpolyribonucleotide of claim 15, wherein each spacer region is from 5 to500 ribonucleotides in length.
 17. The linear polyribonucleotide ofclaim 1, wherein the linear polyribonucleotide is at least 1,000ribonucleotides in length.
 18. The linear polyribonucleotide of claim17, wherein the linear polyribonucleotide is at least 3,000ribonucleotides in length.
 19. The linear polyribonucleotide of claim 1,wherein the polyribonucleotide cargo is at least 1,000 ribonucleotidesin length.
 20. The linear polyribonucleotide of claim 19, wherein thepolyribonucleotide cargo is at least 3,000 ribonucleotides in length.21. A circular polyribonucleotide produced from the linearpolyribonucleotide of claim
 1. 22. A method of expressing a polypeptidein a cell, the method comprising providing the linear polyribonucleotideof claim 1 to the cell, wherein the polyribonucleotide cargo comprisesan expression sequence encoding the polypeptide.
 23. A method ofproducing a circular polyribonucleotide, the method comprising providingthe linear polyribonucleotide of claim 1 under conditions suitable forself-splicing of the linear polyribonucleotide to produce the circularpolyribonucleotide.